The Pennsylvania State University

The Graduate School

College of Medicine

AN INVESTIGATION OF MU-OPIOID RECEPTOR INTERACTING PROTEINS

WITHIN THE MU-OPIOID RECEPTOR SIGNALING COMPLEX

A Dissertation in

Genetics

by

Stephanie Justice-Bitner

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Doctor of Philosophy

December 2012

The dissertation of Stephanie Justice-Bitner was reviewed and approved* by the following:

Robert Levenson

Distinguished Professor of Pharmacology

Co-Director of MD/PhD Program

Dissertation Advisor

Chair of Committee

Laura Carrel

Associate Professor of Biochemistry and Molecular Biology

Vincent Chau

Professor of Cellular and Molecular Physiology

Victor Ruiz-Velasco

Associate Professor of Anesthesiology

David Spector

Professor and Distinguished Educator of Microbiology and Immunology

Co-chair, Intercollege Graduate Program in Genetics

*Signatures are on file in the Graduate School

iii

ABSTRACT

Opioids are powerful analgesic drugs that lead to the development of tolerance, physical

dependence, and addiction with prolonged use or abuse. Chronic exposure appears to cause

alterations in the neuronal morphology of brain regions implicated in opioid dependence and

addiction. Many of these changes reflect the inhibitory effects of these drugs and other opioid

receptor agonists on neuron size, neurite outgrowth, dendritic arborization, and neurogenesis that

occur in brain regions important for reward processing, learning, and memory. The mu-opioid

receptor (MOR) is the G-protein coupled receptor (GPCR) primarily responsible for mediating

the analgesic and rewarding properties of opioid agonist drugs such as morphine, fentanyl, and

heroin. However, the precise role of the MOR in the development of these neuronal alterations

remains elusive. A growing body of evidence suggests that G-protein-coupled receptor (GPCR)

signaling is modulated by proteins that bind GPCRs and form multiprotein signaling complexes.

A number of proteins that interact with the MOR have recently been identified and have been

shown to affect MOR biogenesis, trafficking, and signaling. Therefore, identifying and

characterizing MOR interacting proteins (MORIPs) may help to elucidate the underlying

mechanisms of opioid addiction.

Using a combination of conventional and modified membrane yeast two-hybrid (MYTH)

screening methods, a cohort of novel MOR interacting proteins (MORIPs) was identified. The

interaction between the MOR and a subset of MORIPs was validated in pull-down, co-

immunoprecipitation, and co-localization studies using HEK293 cells stably expressing the MOR

as well as rodent brain. Additionally, a subset of MORIPs was found capable of interaction with

the delta (DOR) and kappa (KOR) opioid receptors. Finally, three of these proteins showed

altered expression in the brains of morphine treated mice.

The ubiquitin pathway has been shown to be altered in the addiction process, therefore

iv

two novel MORIPs, seven in absentia 1(SIAH1) and 2 (SIAH2), were of particular interest due to

their known function as E3 ligases in the ubiquitin pathway. The effects of altering SIAH1

and/or SIAH2 protein expression on opioid receptor ubiquitination and protein levels was

investigated using human embryonic kidney (HEK) stably transfected with MOR, delta opioid

receptor (DOR), or kappa opioid receptor (KOR). These experiments suggest that SIAH2 is an

E3 ligase for the MOR, while both SIAH1 and SIAH2 may conjugate ubiquitin to DOR and

KOR. The ubiquitination by SIAH2 appears to signal for degradation of the MOR, whereas

SIAH1 seems to act as a shuttle protein or chaperone assisting in degradation. In contrast, the

ubiquitination of DOR and KOR may be a signal for degradation or trafficking of receptors.

Additionally, opioid agonist treatment of neuroblastoma cells promoted the formation of

MOR/SIAH1 complexes. Based on the known function of these newly identified MORIPs, the

interactions forming the MOR signalplex are hypothesized to be important for MOR signaling

and intracellular trafficking. Understanding the molecular complexity of MOR/MORIP

interactions provides a conceptual framework for defining the cellular mechanisms of MOR

signaling in brain and may be critical for determining the physiological basis of opioid tolerance

and addiction.

v

TABLE OF CONTENTS

LIST OF FIGURES ................................................................................................................. viii

LIST OF TABLES ................................................................................................................... x

LIST OF COMMON ABBREVIATIONS .............................................................................. xi

ACKNOWLEDGEMENTS ..................................................................................................... xiii

Chapter 1 Literature Review .................................................................................................... 1

1.1 Opioid Drugs .............................................................................................................. 1 1.1.1 Analgesia and Addiction ................................................................................. 1 1.1.2 Opioid Pharmacology ...................................................................................... 3

1.2 The Opioid System ..................................................................................................... 5 1.2.1 The Opioid Receptors ...................................................................................... 6 1.2.3 Mu-Opioid Receptor Genetic Variations ........................................................ 8 1.2.4 Dimerization of Opioid Receptors .................................................................. 17 1.2.5 Endogenous Opioid Peptides .......................................................................... 19

1.3 Neurobiology of Opioid Exposure ............................................................................. 21 1.3.1 Mechanisms of Opioid Addiction ................................................................... 21 1.3.2 Animal Models of Addiction-Like Behavior .................................................. 22 1.3.3 Opioid Receptor Knockout Studies ................................................................. 23 1.3.4 Physiological and Psychological Adaptations to Opioid Exposure ................ 26 1.3.5 Regulation of MOR ......................................................................................... 29

1.4 GPCR Interacting Proteins ......................................................................................... 31 1.4.1 MOR Interacting Proteins ............................................................................... 32 1.4.2 Identifying Candidate MORIPs ....................................................................... 35

1.5 Rationale and Hypothesis ........................................................................................... 36

Chapter 2 Identification of MOR Interacting Proteins ............................................................. 38

2.1 Introduction ................................................................................................................ 38 2.2 Experimental Procedures ........................................................................................... 40

2.2.1 Membrane Yeast Two-Hybrid Screen ............................................................. 40 2.2.2 Conventional Yeast Two-Hybrid Screen ........................................................ 41 2.2.3 Mapping of Binding Regions .......................................................................... 42 2.2.4 Glutathione S-transferase Pulldown Assay ..................................................... 42 2.2.5 Cell Culture ..................................................................................................... 44 2.2.6 Co-immunoprecipitation and Western blot Analysis ...................................... 45 2.2.7 Immunofluorescence Microscopy ................................................................... 46

2.3 Results ........................................................................................................................ 46 2.3.1 Identification of Novel MOR Interacting Proteins .......................................... 46 2.3.2 Mapping of Binding Regions .......................................................................... 51 2.3.3 Validation of MOR/MORIP interaction by GST Pulldown ............................ 53

vi

2.3.4 Interaction of MORIPs with MOR, DOR, and KOR in Cultured

Mammalian Cells ............................................................................................. 55 2.3.5 Immunofluorescence Microscopy ................................................................... 57

2.4 Discussion .................................................................................................................. 59

Chapter 3 Functional Analysis of the MOR/SIAH Interaction ................................................ 64

3.1 Introduction ................................................................................................................ 64 3.2 Experimental Procedures ........................................................................................... 68

3.2.1 Mapping of binding sites ................................................................................. 68 3.2.2 Cell culture and transfections .......................................................................... 69 3.2.3 Treatment of cells with inhibitors of the proteasome and lysosome ............... 70 3.2.4 Detection of ubiquitinated MOR using TUBEs .............................................. 70 3.2.5 siRNA knockdown of SIAH proteins .............................................................. 71 3.2.6 Co-immunoprecipitation and western blot analysis ........................................ 71 3.2.7 Quantitation of Western blots ......................................................................... 73

3.3 Results ........................................................................................................................ 73 3.3.1 Mapping of the MOR binding site on SIAH1 ................................................. 73 3.3.2 SIAH1 regulates the amount of MOR modified with ubiquitin ...................... 75 3.3.3 siRNA knockdown of SIAH1 increases the ubiquitination of the opioid

receptors ........................................................................................................... 78 3.3.4 Knockdown of SIAH2 results in decreased ubiquitination of MOR, but

increased ubiquitination of DOR and KOR. ..................................................... 81 3.3.5 The MOR undergoes degradation via the proteasome and lysosome ............. 83 3.3.6 Overexpression of SIAH1 causes a decrease in MOR protein levels.............. 85 3.3.7 siRNA knockdown of SIAH1 increases MOR protein levels, but

decreases DOR and KOR protein ..................................................................... 86 3.3.8 siRNA knockdown of SIAH2 decreases MOR protein levels, but

increases DOR and KOR protein ..................................................................... 89 3.3.9 SIAH1 and SIAH2 interact in HEK-MOR cells.............................................. 91 3.3.10 SIAH1 regulates the levels of SIAH2 in HEK cells ...................................... 92 3.3.11 Knockdown of both SIAH1 and SIAH2 results in a decrease in the

ubiquitination of the MOR and an increase in MOR protein levels ................. 94 3.4 Discussion .................................................................................................................. 96

Chapter 4 Effect of Opioid Exposure on MORIPs .................................................................. 104

4.1 Introduction ................................................................................................................ 104 4.2 Experimental Procedures ........................................................................................... 107

4.2.1 Cell culture and drug treatment ....................................................................... 107 4.2.2 Co-immunoprecipitation and Western blot analysis ....................................... 108 4.2.3 Animal Care and Drug Treatment ................................................................... 109 4.2.4 Tissue Preparation and Protein Analysis ......................................................... 110

4.3 Results ........................................................................................................................ 111 4.3.1 Drug exposure alters strength of interaction between SIAH1 and opioid

receptors ........................................................................................................... 111 4.3.2 Interaction of MOR and MORIPs in Rodent Brain ......................................... 114 4.3.3 The Effect of Morphine on MORIP Expression ............................................. 117

4.4 Discussion .................................................................................................................. 119

vii

Chapter 5 Overall Discussion .................................................................................................. 123

REFERENCES ........................................................................................................................ 140

viii

LIST OF FIGURES

Figure 1.1: Non-selective agonists and antagonists at opioid receptors .................................. 5

Figure 1.2: Structural similarity between opioid receptors ...................................................... 7

Figure 1.3: Mouse strains show varied responses to morphine and other opioid drugs .......... 10

Figure 1.4: Schematic of the human OMPR1 gene structure and splice variants .................... 13

Figure 2.1: Mapping of MORIP binding regions ..................................................................... 52

Figure 2.2: GST pull down of MORIPs and MOR .................................................................. 54

Figure 2.3: Co-immunoprecipitation of MORIPs and MOR, KOR, and DOR ........................ 56

Figure 2.4: Co-localization of MOR and MORIPs .................................................................. 59

Figure 3.1: Mapping of SIAH1 binding site ............................................................................ 74

Figure 3.2: SIAH1 decreases the ubiquitination of the MOR .................................................. 77

Figure 3.3: Knockdown of SIAH1 increases ubiquitination of MOR, DOR, and KOR .......... 80

Figure 3.4: Knockdown of SIAH2 decreases ubiquitination of MOR, but increases

ubiquitination of DOR and KOR ..................................................................................... 82

Figure 3.5: Inhibitor treatment of HEK-MOR cells ................................................................. 85

Figure 3.6: SIAH1 induces MOR degradation......................................................................... 86

Figure 3.7: Knockdown of SIAH1 changes MOR, DOR, and KOR protein levels ................. 88

Figure 3.8: Knockdown of SIAH2 changes MOR, DOR, and KOR protein levels ................. 90

Figure 3.9: Co-immunoprecipitation of SIAH1 with SIAH2................................................... 92

Figure 3.10: SIAH1 knockdown causes an increase in SIAH2 protein levels ......................... 93

Figure 3.11: Knockdown of SIAH2 does not alter SIAH1 protein levels................................ 94

Figure 3.12: Knockdown of both SIAH1 and SIAH2 decreases MOR ubiquitnation and

increases MOR protein levels .......................................................................................... 96

Figure 4.1: DAMGO treatment of SH-SY5Y cells promotes MOR/SIAH1 complex

formation .......................................................................................................................... 112

Figure 4.2: Morphine treatment promotes DOR/SIAH1 complex formation .......................... 114

ix

Figure 4.3: MORIP expression and interaction with MOR in mouse brain ............................. 116

Figure 4.4: MORIP expression in brain regions of morphine treated mice ............................. 118

Figure 5.1: MOR interaction map ............................................................................................ 124

Figure 5.2: Models for SIAH1 and SIAH2 function in HEK-DOR and HEK-MOR cells ...... 130

Figure 5.3: Simplified model of Siah protein-mediated MOR degradation ............................. 132

Figure 5.4: Model of the role of SIAH1 and SIAH2 in ER-associated degradation of the

MOR ................................................................................................................................ 137

x

LIST OF TABLES

Table 1.1: Legal Definitions of Schedule I and Schedule II Controlled Substances ............... 2

Table 1.2: Association studies investigating A118G SNP in opioid dependence in

humans ............................................................................................................................. 16

Table 1.3: Opioid receptor dimers with related G protein-coupled receptors .......................... 18

Table 1.4: Endogenous opioid peptides ................................................................................... 20

Table 1.5: Summary of opioid receptor knockout mice studies............................................... 26

Table 1.6: Previously identified MORIPs ................................................................................ 33

Table 2.1: PCR primers used for cloning MORIPs ................................................................. 44

Table 2.2: Putative MORIPs identified in MYTH screen ........................................................ 48

Table 2.3: Putative MORIPs identified in conventional yeast two-hybrid screens ................. 50

Table 3.1: PCR primers used for cloning of SIAH1 deletion mutants ..................................... 69

Table 3.2: Summary of SIAH overexpression and knockdown experiments .......................... 99

xi

LIST OF COMMON ABBREVIATIONS

AUP1 ancient ubiquitous protein 1

bp base pairs

coIP co-immunoprecipitation

CSN5 COP9 signalosome subunit 5

DOK4 docking protein 4

DOK5 docking protein 5

DOR δ-opioid receptor

ECL enhanced chemiluminescence

FBS fetal bovine serum

GPCR G protein-coupled receptor

GST glutathione S-transferase

HEK human embryonic kidney

HRP horseradish peroxidase

IL intracellular loop

IP immunoprecipitation

kDa kilodalton

KO knockout

KOR κ-opioid receptor

MOR μ-opioid receptor

MORIP μ-opioid receptor interacting protein

MYTH membrane yeast two-hybrid

PAGE polyacrylamide gel electrophoresis

xii

PBS phosphate buffered saline

PVDF polyvinylidine difluoride

SIAH1 seven in absentia homolog 1

SIAH2 seven in absentia homolog 2

SDS sodium dodecyl sulfate

SNP single nucleotide polymorphism

VAPA vesicle-associated membrane protein-A

VTA ventral tegmental area

WLS Wntless

Y2H yeast two-hybrid

xiii

ACKNOWLEDGEMENTS

I am forever grateful to all of those who have contributed to the completion of my

graduate work. First, I would like to thank Dr. Robert Levenson, my thesis advisor, for his

support, encouragement, and patience. I am grateful to the members of my thesis committee, Drs.

Vincent Chau, Victor Ruiz-Velasco, and Laura Carrel for their advice and support throughout my

graduate school career. I especially thank Dr. Vincent Chau for his technical advice and support.

I would also like to thank the past and present members of the Levenson Lab, who have made the

lab an enjoyable place to work, especially Dr. Jessica Petko for her unending encouragement,

advice, and friendship.

I extend gratitude to my collaborators, including Drs. Igor Staljar, Tom Ferraro, and

Jessica Petko for their assistance and advice. I am also indebted to the faculty and staff of the

Pharmacology Department and the Genetics Program.

Finally, I want to acknowledge my family members. I thank my parents for their

continued love and support throughout my schooling. I thank my husband’s aunt for providing

me a place to live, while working on my research. Lastly, I thank my husband for having the

patience to care for our dogs and home miles away while I worked on my graduate work.

Chapter 1

Literature Review

1.1 Opioid Drugs

1.1.1 Analgesia and Addiction

Opioid analgesics are invaluable in the treatment of severe, chronic, and/or

unremitting pain symptoms. The effectiveness of these drugs in relieving pain has been

known for hundreds of years (Dhawan et al., 1996). Unfortunately, these pain-relieving

benefits come with a number of negative side effects, which limits their use for only the

most severe pain conditions. The major side effects of opioid analgesics include

respiratory depression, alterations in heart rate and blood pressure, severe constipation,

and mood changes. The most significant of these effects are respiratory depression and

mood changes. The most common cause of death in cases of opioid agonist overdose is

respiratory depression. The reported feelings of euphoria and relaxation that typically

occur shortly after administration of opioid agonist drugs have been implicated in the

abuse liability of these drugs. Psychologically, this euphoric state is considered to be the

motivating or rewarding factor that drives an individual to use these drugs inappropriately

(Bailey and Connor, 2005; Corbett et al., 2006).

Due to their potential for abuse, opioid agonist drugs fall under the auspices of the

Controlled Substances Act, and are considered either Schedule I or Schedule II controlled

substances depending on whether or not the drug is clinically useful (Table 1.1). For

example, heroin (diacetylmorphine) has been deemed to have no medical use in the

United States, and is therefore listed as a Schedule I controlled substance. On the other

hand, morphine is considered to have an important medical use and is therefore listed as a

Schedule II controlled substance. The existence of the Controlled Substances Act affirms

2

the larger problem that these drugs pose from a biological, societal, and legislative

perspective.

Table 1.1: Legal definitions of Schedule I and Schedule II controlled substances

These definitions were reproduced verbatim from Section 812 of the Controlled

Substances Act that was enacted into law by the Congress of the United States as Title II

of the Comprehensive Drug Abuse and Prevention and Control Act of 1970. It should be

noted that three more schedules exist, but only one opioid mixed agonist/antagonist,

nalorphrine, is schedule III. Schedule III drugs have less potential for abuse than

schedule I and II, but can still cause substance dependence.

Schedule I

(A) The drug or other substance has a high potential for abuse.

(B) The drug or other substance has no currently accepted

medical use in treatment in the United States.

(C) There is a lack of accepted safety for use of the drug or

other substance under medical supervision.

Schedule II

(A) The drug or other substance has a high potential for abuse.

(B) The drug or other substance has a currently accepted

medical use in treatment in the United States or a currently

accepted medical use with severe restrictions.

(C) Abuse of the drug or other substances may lead to severe

psychological of physical dependence.

In the 2010 National Survey on Drug Use and Health, it was estimated that 22.6

million Americans 12 years or older had used an illicit drug or used a prescription drug

inappropriately in the past year. Of these 22.6 million, an estimated 9.0 million people

3

were current users of drugs other than marijuana and 5.3 million of these current drug

users were using prescription pain killers (opioid drugs) or heroin. Opioid drugs are the

third most abused drug behind alcohol and marijuana. The use of prescription opioid

drugs among first-time initiators of drug abuse was found to be almost equal to

marijuana. However, the ability of many users to obtain opioid drugs from a friend or

relative for free could lead to an increase of opioid drug users.

The simplest way to resolve these issues would be the development of an

analgesic drug that is as efficacious as known opioid analgesics, but does not have

potential for abuse. Some researchers have called this endeavor “the exciting but vain

quest for the Holy Grail” (Corbett et al., 2006). When considering the efforts made in the

diverse field of opioid-related research, the sum total of work can all be traced back to

this goal of finding or creating a drug that can alleviate severe pain without being

addictive.

1.1.2 Opioid Pharmacology

Opioid compounds have been used since antiquity; long before the opioid

receptors and their peptide ligands were discovered. It wasn’t until 1805 that the first and

prototypical opioid agonist, morphine, was isolated from opium. Morphine is invaluable

as an analgesic agent, but it can induce a sense of euphoria, which contributes to non-

medical use and abuse (Dhawan et al., 1996). Receptor knockout studies in mice have

shown that both the analgesic and rewarding properties of morphine are mediated by the

mu-opioid receptor (MOR) (Matthes et al., 1996). Since these drugs are such powerful

analgesics, much effort has gone into synthesizing new drugs that retain the pain-

relieving qualities of morphine, but are less rewarding.

4

The quest to find a better opioid drug has led to the development of a large library

of compounds. This library consists of substances that act as agonists, antagonists, and

mixed agonist-antagonists. Morphine is the archetypal opioid agonist that mediates its

effects by binding to and activating opioid receptors. Antagonists, such as naloxone and

naltrexone, block the effects of agonists by competing for receptor binding sites and

preventing activation of receptors by agonists. Antagonists are useful in clinical settings

to rapidly reverse the effects of opioid agonist overdoses. Mixed agonist-antagonists are

compounds, such as nalorphine (n-allylnormorphine), which can act as an agonist at one

opioid receptor subtype, but also act as an antagonist at another opioid receptor subtype.

The development of these mixed action drugs initially held the promise of promoting one

effect (analgesia) while controlling another (addiction), but these drugs have still had

enough abuse liability to remain problematic (Snyder, 2004).

Most of these opioid drugs were created by making small chemical modifications

to morphine. Morphine is a naturally occurring plant alkaloid isolated from opium,

which is harvested from the unripe seed capsule of the poppy plant, Papaver somniferum

(Dhawan et al., 1996). When the structure of morphine and naloxone is compared, all

that is required to convert a potent agonist into an antagonist is an ethylene side-chain

(Figure 1.1). Although this effect is not fully understood, the fact that small differences

in chemical structure can have large impacts on pharmacological activity is clear from

studies that show that the opioid binding sites in the brain are stereospecific (Simon et al.,

1973; Terenius, 1973).

5

Figure 1.1: Non-selective agonists and antagonists at opioid receptors

Each of the compounds above contains the pentacyclic structure of morphine. This basic

structure contains a benzene ring with a phenolic hydroxyl group at position 3 and an

alcohol hydroxyl group at position 6 and at the nitrogen atom. The addition of relatively

simple side chains to this basic structure results in the altered activity of the resulting

compound. Reproduced from (Dhawan et al., 1996).

1.2 The Opioid System

Given the centuries-old knowledge and use of opioid drugs, it is surprising that

the existence of an endogenous opioid receptor was not postulated until the 1950s

(Lasagna, 1954). Since that time, pharmacological studies (Martin et al., 1976) and the

cloning of specific genes have led to the identification of the components of the

endogenous opioid system (Kieffer and Gaveriaux-Ruff, 2002).

6

The endogenous opioid system is composed of a subfamily of G-protein coupled

receptors (GPCRs) and their peptide ligands, most of which are derived from precursor

proteins. Many important physiological functions are regulated by this system, including

respiration, gastrointestinal motility, hormone secretion, thermoregulation, immune

function, mood, and pain modulation (Dhawan et al., 1996). The anatomical expression

profile of opioid receptors and ligands throughout the central and peripheral nervous

system and in other parts of the body, such as the gastrointestinal tract, explains why this

system is involved in so many physiological functions (Snyder, 2004; Snyder and

Childers, 1979). Our knowledge of the endogenous opioid system is largely derived from

the major effects and side effects that are seen when exogenous opioid drugs act on the

endogenous receptors (Dhawan et al., 1996; Snyder, 2004).

1.2.1 The Opioid Receptors

There are four main subtypes of endogenous opioid receptors, which are

designated delta (DORkappa (KOR), mu (MOR), and nociceptin (NOR). All four

receptors belong to the Class A (rhodopsin-like) GPCRs, and share similar structural

features, including an extracellular amino-terminus, seven transmembrane spanning

domains linked by three extracellular and intracellular loops, and an intracellular

carboxy-terminus. The opioid receptors are often posttranslationally modified through

glycosylation and palmitoylation (Minami and Satoh, 1995), and exert their major

downstream effects through pertussis toxin-sensitive heterotrimeric G-proteins (Gαo)

(Johnson et al., 1994; Ueda et al., 1988; Wong et al., 1988). Each receptor is encoded by

a single gene in humans and presumably forms a single gene product (Knapp et al., 1994;

Wang et al., 1994; Zhu et al., 1995), although a number of splice variants have been

reported for the MOR (Pasternak, 2004). Amino acid sequence alignments show that the

receptors are homologous to each other (MORand KOR 68% identical, MOR and DOR

66% identical, KOR and DOR 58% identical; NOR shares 50-60% identity with DOR,

, and MOR) with most of the variation between receptors occurring in the

7

extracellular loops and the carboxy-terminus (Dhawan et al., 1996). Figure 1.2

summarizes the structural similarities and differences between the opioid receptor types.

Recombinant chimeric receptor studies have found that the extracellular regions are

responsible for the specificity of ligand binding (Kong et al., 1994; Onogi et al., 1995;

Xue et al., 1994). Additionally, single nucleotide polymorphisms can alter ligand

binding. For example, an amino acid substitution at residue 40 (Asn to Asp) of the MOR

has been shown to increase -endorphin binding affinity (Bond et al., 1998) and alters the

functional properties of the receptor (Kroslak et al., 2007).

Figure 1.2: Structural similarity between opioid receptors

Model for the mu-opioid receptor in the rat. Amino acid residues that are conserved

between the mu-opioid receptor and both delta- and kappa- opioid receptors are shown in

black. Gray indicates an amino acid conserved with two opioid receptors and white

indicates non-conserved residues. Branched structures indicate potential N-linked

glycosylation sites. Reproduced from Minami and Satoh, 1995.

8

The distribution of opioid receptors has been reviewed by Le Merrer et al (2009)

and will not be addressed in detail in this chapter. Opioid receptors are primarily

expressed in the cortex, limbic system, and the brain stem. Ligand binding studies have

shown that the three opioid receptors exhibit an overlapping expression pattern in most

brain structures; however, some structures exhibit a higher expression of one receptor

over the others. For example, the MOR is more highly expressed in the thalamus when

compared to either the DOR or the KOR. Interestingly, MORs and KORs are often

found together in most brain regions, whereas the distribution of DORs is more restricted.

Importantly, all three receptors have been shown to have expression in areas of the brain

believed to be involved with addiction, such as the hippocampus, ventral tegmental area,

and the nucleus accumbens (Le Merrer et al., 2009). The distribution of opioid receptors

also correlates with the reported functions of the opioid system. For example, activation

of MORs located in the periaqueductal grey area (PAG) leads to analgesia, while

activation of NORs in the same area reduces the analgesic effect of MOR agonists. This

difference is due to the location of these receptors within the PAG. MORs are located on

presynaptic inhibitory interneurons, which leads to disinhibition of the neural output from

the PAG, whereas NORs are located on postsynaptic neurons, which leads to inhibition

of the neural output from the PAG. This anatomical difference explains the opposing

effects of these two opioid receptors, despite the fact that both receptors are coupled to

the same downstream effectors (Corbett et al., 2006). Knockout studies in mice have

further confirmed the role of these receptors in pain modulation, reward, and behavior

(Kieffer and Gaveriaux-Ruff, 2002).

1.2.3 Mu-Opioid Receptor Genetic Variations

Opioid agonists used in the treatment of pain have a narrow therapeutic index and

large interpatient variability in response. Patients may respond better to one opioid than

another with respect to both analgesia and side effects (Pasternak, 2004). Even in

relatively homogeneous patient cohorts, dosage requirements can vary substantially. For

example, in a study of postoperative hip replacement therapy encompassing over 3,000

9

patients, morphine dosage requirements varied almost 40-fold (Aubrun et al., 2003).

There also is great variability in the responses to these drugs, which may lead to the

development of addiction (Trigo et al., 2010; Volkow and Li, 2005). Similar

observations have also been seen in animal models (Figure 1.3). The analgesic

sensitivity of various strains of mice to a fixed morphine dose was observed to vary

substantially, with BALB/c mice the most sensitive and CXBK mice being the least

sensitive (Picker et al., 1991). When similar doses of opioids were given to CD-1 and

CXBK mice, there were more varied differences in analgesia depending on the opioid

drug administered (Figure 1.3) (Chang et al., 1998; Rossi et al., 1996). In addition to

inbred mouse strains, inbred rat strains have also shown variation in response to opioid

drugs. Among these inbred strains, Lewis and Fischer rats are the most often compared

because of their behavioral and neuronal molecular differences in responding to drugs.

Lewis rats tend to be more responsive in behavioral tests because they more easily self-

administer morphine and other drugs, such as cocaine (Ambrosio et al., 1995). In a more

recent study, Lewis rats were found to escalate both the dose and total amount of heroin

administered, whereas Fischer rats did not escalate either (Picetti et al., 2012). These

observed varied responses in animal models and in human subjects are believed to be

mediated by splice variants and polymorphisms in the MOR (Kasai and Ikeda, 2011).

10

Figure 1.3: Mouse strains show varied responses to morphine and other opioid

drugs

These two graphs represent some of the mouse strain studies that have been reported in

the literature. (A) The strains of mice received a fixed dose of morphine and were

assessed for analgesia using the radiant heat tailflck assay (Pick et al., 1991). (B) Doses

of opioids were chosen to give similar responses between CD-1 mice. These same doses

were then administered to CXBK mice (Rossi et al., 1996; Chang et al., 1998). Images

reproduced from Paskernak, 2004.

There are at least three different categories of factors that contribute to the

vulnerability of developing addiction: environmental factors, such as cues and

conditioning; drug-induced factors, which lead to molecular and neurobiological changes;

and genetic factors, which represent 40-60% of the risk for developing addiction (Kreek

et al., 2005). Included in these genetic factors are opioid receptor splice variants and

A

B

11

single nucleotide polymorphisms (SNPs), both of which have been studied in animal

models and in humans.

Data from animal models has provided important information regarding the role

of genetic factors in addiction. In mu-opioid receptor knockout mice, homozygous mice

have lower nociceptive thresholds than heterozygous knockout mice that have 50% less

wild-type receptor density. In turn, these heterozygous mice have lower nociceptive

thresholds than wild-type mice with normal mu-opioid receptor densities (Sora et al.,

1997). Mouse-strain comparison studies in recombinant inbred mouse lines have

provided additional evidence to implicate genetic factors in addiction. The CXBK mouse

strain exhibited reduced responses to some opioid agonists. Like the heterozygous MOR

knockout mice, CXBK mice express approximately 50% less MOR compared with

progenitor strains (BALB/c and C57BL/6) and have a similar phenotype (Ikeda et al.,

1999; Ikeda et al., 2001).

The human MOR gene (OPRM1) spans over 200 kilobases and consists of 11

exons that combine to yield splice variants (Xu et al., 2009). The most abundant

transcript, MOR-1, which consists of exons 1, 2, 3, and 4, is approximately 15 kb in

length, and encodes 400 amino acids (Ide et al., 2005). Mouse MOR-1 shares 94%

amino acid identity to human MOR-1. The first splice variant to be discovered was

human MOR-1A. This variant was shown to encode for only 392 amino acids and differs

from MOR-1 only at its 3’end (Bare et al., 1994). Since this initial discovery, over 28

splice variants from the mouse OPRM1 gene (Doyle et al., 2007a; Doyle et al., 2007b;

Pan, 2005) have been isolated with similar splicing patterns observed in rats (Pasternak et

al., 2004) and humans (Pan, 2005; Pan et al., 2003). The majority of these splice variants

yield receptors that differ from each other at the C-terminus. Splicing typically occurs

between exon 3 and more than 10 downstream exons, yielding at least 16 carboxyl

terminal variants (Figure 1.3). Although these full-length variants share a common

binding pocket (as seven transmembrane domains are conserved), they display significant

differences in mu opioid receptor agonist-induced G protein activation (Bolan et al.,

2004; Oldfield et al., 2008; Pan et al., 2005; Pasternak et al., 2004). These studies used

[35

S] GTPγS binding assays (measures G-protein activation) to show that the potency and

12

efficacy of opioid agonists, such as DAMGO and morphine, differed among splice

variants. Additionally, these variants have different distributions in the types of cells and

regions of the central nervous system (Abbadie et al., 2000a; Abbadie et al., 2000b;

Abbadie et al., 2000c; Abbadie et al., 2001; Schnell and Wessendorf, 2004). For

example, in the dorsal horn of the spinal cord, cells express either MOR-1 or MOR-1C,

but not both together (Pasternak, 2005).

In addition to the 3’end splice variants described above, there are also 5’ end

splice variants that contain exon 11. This exon is located 28-30 kb upstream from exon

1(species dependent). To date, nine exon 11-associated splice variants have been

identified (Xu et al., 2009). Interestingly, three of the transcripts generate the same

protein as MOR-1, while others predict a protein with six transmembrane domains,

and/or a protein with one transmembrane domain (Pan et al., 2001). These variants also

show differing expression patterns in the brain (Abbadie et al., 2004). Recently, a

naltrexone derivative, iodobenzoylnaltrexamide (IBNtxA), was synthesized in the

Pasternak laboratory (Majumdar et al., 2011). This new opioid drug is a potent analgesic,

but lacks the common opioid side effects, such as respiratory depression, constipation, or

physical dependence. In studying this new drug, Pasternak and colleagues found that

IBNtxA analgesia persisted in triple knockout mice (no MOR-1, DOR, or KOR). In

contrast, there was a loss of both IBNtxA analgesia and receptor binding in exon 11

knockout mice (Majumdar et al., 2011). These results demonstrate that exon splice

variants may be of interest in designing opioid drugs with analgesic power and little

abuse liability.

13

Figure 1.4: Schematic of the human OMPR1 gene structure and splice variants

Exons are represented by boxes, while introns are represented by horizontal lines.

Translational start sites are indicated by downward lines on exon boxes. Translation stop

sites are indicated by upward lines on exon boxes. Exons are numbered by the order of

their isolation and/or similarity to exons identified in the mouse. C-terminal splice

variants would include MOR-1A, MOR-1B-1B5, MOR-1O, MOR-1S, MOR-1X, MOR-

1Y, Mu3, SV1, and SV2. Exon 11 splice variants would include MOR-1G1, MOR-1G2,

MOR-1H, and MOR-1i. Reproduced from Xu et al., 2009.

14

According to the dbSNP database and the NCBI database of genetic variation, the

OPRM1 gene contains over 700 single nucleotide polymorphisms (SNPs). This genetic

variation differs greatly between different races and ethnicities. For example, the minor

allele frequency (MAF) of A118G, which is a well-studied nonsynonymous SNP in exon

1 leading to an Asn40Asp substitution, are 0.047 in the African population, 0.154 in the

European population, 0.485 in the Japanese population, 0.14 in the Hispanic population,

and 0.08 in the Bedouin population (Gelernter et al., 1999). Many SNPs have been

studied to determine their association with pain sensitivity. Four SNPs (G-172T, A118G,

IVS1-C2994T, and IVS2 +G31A) were analyzed in association studies with pain.

Among these SNPs, two (IVS1-C2994T and IVS2 +G31A) were found to be significantly

associated with pain sensitivity scores and pressure pain thresholds, respectively

(Fillingim et al., 2005; Huang et al., 2008; Shabalina et al., 2009). However, there have

been no other reports showing an association between these two SNPs and pain-related

traits. Significant associations have also been observed in studies of the A118G SNP.

The G-allele carriers of the A118G SNP showed higher pressure pain thresholds and pain

tolerance thresholds (Fillingim et al., 2008).

Other than the highly studied A118G SNP, few other polymorphisms have been

investigated for functional significance. Among those that have been studied are two

promoter SNPs, G-554A and A-1320G. G-554A decreases transcription of MOR, but is

rare with a MAF of less than 0.001. In contrast, the A-1320G variant increases

transcription of MOR (Bayerer et al., 2007). Three others, G779A, G794A, and T802C,

are located in exon 3 and cause a decrease in receptor coupling and signaling (Befort et

al., 2001; Koch et al., 2000; Wang et al., 2001). Other OPRM1 polymorphisms have

been identified and associated with either pain or opioid dependence, but have no in vitro

evidence for function. These include C17T (A6V), which is found primarily in African

Americans (Crystal et al., 2012; Hoehe et al., 2000) and C440G (S147C), which is rare

with an MAF of less than 0.006 (Glatt et al., 2007).

The MOR mediates analgesia and side effects, such as nausea and respiratory

depression, of opioid drugs. The analgesic and side effects of opioids were abolished or

reduced in homozygous or heterozygous MOR-deficient mice, indicating that MOR gene

15

dosage is related to the clinical efficacy of these drugs. Although many SNPs in the

OPRM1 gene have been investigated in regard to opioid sensitivity, the one most often

studied is A118G. The A118G SNP has been shown to be associated with the analgesic

and side effects of opioids. In these studies, opioid dose (Ginosar et al., 2009; Reyes-

Gibby et al., 2007) and consumption (Lotsch et al., 2002; Sia et al., 2008) were greater in

G-allele carriers when compared to AA individuals. G-allele carriers exhibited a lower

analgesic efficacy and a lower incidence of side effects than AA individuals (Kasai and

Ikeda, 2011; Mague and Blendy, 2010; Somogyi et al., 2007). However, other studies

have found no such associations (Kasai and Ikeda, 2011; Mague and Blendy, 2010). The

A118G SNP has been studied in a variety of substance abuse studies, including those for

alcohol, nicotine, heroin, and alcohol. Findings differ greatly from study to study (Kasai

and Ikeda, 2011; Somogyi et al., 2007). As opioid dependence is the focus of this work,

a summary of the studies on the A118G allele and opioid dependence are shown in Table

1.2.

Many OPRM1 gene variations have been identified and analyzed for their

associations with pain sensitivity, opioid sensitivity, and susceptibility to drug

dependence. These studies have revealed significant associations between genetic

variations with regard to opioid sensitivity and vulnerability to substance dependence.

However, not every analysis finds a significant association; therefore, the significance of

variations in the OPRM1 gene is still debatable. More research into the mechanisms

underlying MOR expression and function are required to settle this debate.

16

Table 1.2: Association studies investigating A118G SNP in opioid dependence in

humans

This table summarizes association studies done with the A118G SNP and heroin

dependence. Risk or protection is associated with having at least one copy of the SNP.

These studies indicate that A118G SNP can have varied outcomes depending on the race

or ethnicity studied and the sample size. Adapted from (Mague and Blendy, 2010).

Effect Finding Population

(number of subjects)

Reference

Risk G118 associated with

heroin dependence

Swedish (139 heroin-

dependent, 149 control)

(Bart et al.,

2004)

Risk 90% of G118 were

heroin users

European Caucasian (118) (Drakenberg et

al., 2006)

Risk Higher frequency of

G118 in opioid

dependent subjects

Indian (126 opioid-

dependent and 156 control)

(Kapur et al.,

2007)

Risk G118 associated with

heroin dependence

Chinese men (200 heroin

dependent and 97 control)

(Szeto et al.,

2001)

Protective A118 associated with

heroin in Indian, but

not East Asian

populations

Indian (20 dependent, 117

control), Malaysian (25

dependent, 131 control),

Chinese (52 dependent,

156 control)

(Tan et al.,

2003)

Protective

A118 associated with

heroin dependence in

Hispanics

African American (46

dependent, 16 controls),

Caucasian (60 dependent,

44 controls), Hispanic (116

dependent, 78 controls)

(Bond et al.,

1998)

None No association found Chinese (48 heroin

dependent, 48 controls)

(Shi et al.,

2002)

17

1.2.4 Dimerization of Opioid Receptors

Opioid receptors belong to the G protein-coupled receptor (GPCR) Class A

superfamily. GPCRs can form homo- and heterodimers. Importantly, these dimers can

have distinct pharmacological properties. Therefore, dimerization can be seen as a way

for an organism to increase the functional variety of GPCRs. A large body of evidence

for the existence of GPCRs as dimers and oligomers has accumulated in recent years.

GPCR dimerization has been shown to be a physiological process that modifies receptor

pharmacology and regulates function. Heterodimerization can generate receptors with

characteristics distinct from either single receptor, leading to altered pharmacological

properties (George et al., 2002).

Within the opioid receptor family, DORs have been shown to interact with both

KORs and MORs to form heterodimers (Gomes et al., 2000; Jordan and Devi, 1999). In

the case of interactions between DORs and KORs, the heterodimers were found to have

reduced affinities for highly selective KOR or DOR agonists. Cells co-expressing KOR

and DOR also exhibited synergistic effects on agonist signaling. Additionally,

dimerization was found to affect the trafficking of these receptors, as etorphine-induced

trafficking of DOR was greatly reduced in cells expressing KOR/DOR dimers. Etorphine

causes DOR to be internalized, but when DOR/KOR dimers were expressed, the DOR

remained at the cell surface (Jordan and Devi, 1999). MOR/DOR dimers have also been

demonstrated to have altered selective agonist binding affinities (George et al., 2000). In

cells expressing MOR/DOR dimers, low doses of DOR selective ligands produced a

significant increase in the binding of a MOR selective agonist, which enhanced MOR-

mediated signaling (Gomes et al., 2004). Additionally, MOR/DOR dimers constitutively

recruit beta-arrestin 2, which results in alterations in ERK1/2 signaling (Rozenfeld and

Devi, 2007). More recently, MOR/KOR heterodimers expression in spinal cords of

Sprague-Dawley rats was found to be dependent on the estrous cycle stage of females and

was also less expressed in males (Chakrabarti et al., 2010). Opioid receptors have also

been shown to dimerize with distantly related GPCRs (Table 1.3). These receptor

heterodimers exhibit properties that are quite distinct from those of each individual

18

receptor. Unfortunately, the influence of SNPs on heterodimers formation has not been

studied. This lack of study is likely due to the lack of data on SNPs in other receptors

and the functional consequences of these differences.

Table 1.3: Opioid receptor dimers with related G protein-coupled receptors

This table lists the known opioid receptor heterodimers that have been discovered and the

proposed function of the interaction.

Dimer Function Reference

MOR and serotonin

receptor 5-HT(1A)

MOR-induced ERK1/2

phosphorylation was desensitized

with 5-HT(1A) activation

(Cussac et al., 2012)

MOR and α2A-adrenergic Activation of either receptor leads

to an increase in signaling, but

activation of both leads to a

decrease in signaling

(Jordan et al., 2003)

Apelin receptor and KOR Agonist stimulation of either

receptor resulted in higher

ERK1/2 phosphorylation

(Li et al., 2012)

Chemokine CXCR2 and

DOR

Antagonists of CXCR2 enhanced

the effects of DOR agonists

(Parenty et al., 2008)

Opioid receptor-like 1 and

MOR

Reduced potency of mu-agonist to

inhibit adenylate cyclase

(Wang et al., 2005)

Substance P receptor (NK1)

and MOR

Co-internalization with mu-

agonist treatment

(Pfeiffer et al., 2003)

Somatostatin (SST2A)

receptor and MOR

Mu-agonist treatment induced

phosphorylation and

desensitization of both receptors

(Pfeiffer et al., 2002)

19

1.2.5 Endogenous Opioid Peptides

Endogenous opioid peptides include enkephalins, dynorphins, endorphins, and

nociceptin (Bloom, 1983). Early studies that identified and characterized these peptides

suggested the possibility that they were derived from larger precursor proteins (Snyder

and Childers, 1979), which have now been identified and cloned (Chavkin et al., 1983;

Chretien et al., 1979; Mollereau et al., 1996). The enkephalins, endorphins, and

dynorphins contain the same four amino acid residues at their amino-termini (Try-Gly-

Gly-Phe). This sequence was thought to be necessary for binding to opioid receptors

(Snyder and Childers, 1979); however, more recently identified opioid peptides, such as

endomorphins, do not contain this sequence but still bind opioid receptors (Akil et al.,

1998). The endogenous peptides have not been found to be selective to binding to the

different opioid receptors, but there are differences in their binding affinities for the

opioid receptors (Akil et al., 1998; Dhawan et al., 1996). Table 1.4 lists the endogenous

opioid peptides, their precursor proteins, and their binding affinities.

20

Table 1.4: Endogenous opioid peptides

Precursor proteins and resulting peptides are listed along with the relative differences in

binding affinities of the peptide family for the opioid receptor subtypes. Adapted from

(Corbett et al., 2006).

Precursor Gene Peptide Binding Affinity

Proopiomelanocortin POMC Endorphins

α-endorphin

β-endorphin

γ-endorphin

δ-endorphin

MOR, KOR, DOR

Unknown Unknown Endomorphins

endomorphin-1

endomorphin-2

MOR

Preproenkephalin Penk Enkephalins

met5-enkephalin

leu5-enkephalin

DOR MOR, KOR

Preprodynorphin Pdyn Dynorphins

dynorphin A

dynorphin B

α-neoendorphin

β-neoendorphin

KOR MOR, DOR

Prepronociceptin PPNOC Nociceptin NOR

21

1.3 Neurobiology of Opioid Exposure

Historically, addiction of any kind was thought to be a problem of weak-willed

people. The general consensus was that the addicted person was not trying hard enough

to break his or her bad habit. However, with the sustained scientific efforts of the last 40

years, “addiction” has begun to be recognized as a neuropsychiatric disease.

Unfortunately, much of the social stigma associated with addiction still exists to this day

(Hyman, 2007). Opioid addiction is triggered by a chemical substance that activates a

specific set of receptors in the brain. These same receptors are also acted upon by

endogenous ligands to regulate normal neurobiological processes. Since not all people

that are exposed to opioid drugs develop addiction, it is difficult to conclude if the

activation of these receptors is the sole underlying cause of the disease. Twin, family,

and adoption studies have provided evidence that addiction is a complex genetic disorder

that can also be influenced by environmental factors, such as parental support, home

environment, level of education, and socioeconomic status (Lachman, 2006). This

complexity suggests that there are additional mechanisms that help to determine whether

or not a particular individual will develop addiction to an opioid drug.

1.3.1 Mechanisms of Opioid Addiction

Addiction to opioids is thought to be mediated by activation of MORs in areas of

the brain that mediate reward, such as the ventral tegmental area (VTA) and the nucleus

accumbens (NAc), which are the major constituents of the mesolimbic dopaminergic

pathway (Le Merrer et al., 2009). The direct injection of morphine or endomorphins into

these brain areas has been observed to mediate self-administration and conditioned place

preference in animal models (Tang et al., 2005; van Ree and de Wied, 1980; Zangen et

al., 2002). The potency of these exogenous opioid drugs leads to an over-activation of

the dopaminergic pathway that reinforces the drug-taking behavior. Unfortunately, this

explanation only pertains to the psychological and behavioral foundations of opioid

addiction and does not address the biological adaptations that may contribute to this

22

condition. However, these behavioral studies have illustrated the central role of the MOR

in mediating the rewarding effect of these drugs. In a study by David et al. (2008), wild-

type mice and δ-opioid receptor (DOR) knockout mice self-administered morphine

injected into the VTA, but MOR knockout mice did not. Along with the initial

characterization of the MOR knockout mouse (Matthes et al., 1996), these results directly

implicate the MOR as an essential component of the behavioral response to opioid

exposure.

Interestingly, almost all people who receive opioid drugs over a prolonged period

of time will develop tolerance and physical dependence. Although the mechanisms that

underlie the development of these changes are not clear, many studies hypothesize that

the regulation of MORs at the cellular and molecular levels may be involved (Bailey and

Connor, 2005; Harrison et al., 1998).

1.3.2 Animal Models of Addiction-Like Behavior

Animal studies have been crucial in understanding the biology and pathobiology

of drug addiction. In contrast to clinical studies, the subject population can be more

easily controlled for variables. Animal models often focus on the ability of the drugs to

directly control the animal’s behavior. Animal studies have demonstrated that the

rewarding effect is not dependent on preexisting conditions. Exposure to the drug alone

is sufficient to motivate drug-taking behavior (Lynch et al., 2010). There are two major

approaches to assessing addiction-like behavior; conditioned place preference (CPP) and

self-administration.

The CPP procedure exposes animals to a distinct environment in the presence of

drug. If the rodents find this substance positively reinforcing, they will show preference

for that environment when later given a choice (Tzschentke, 1998). CPP is widely used

to study the tolerance and sensitization to the rewarding effects of drugs induced by pre-

treatment regimens. This method can also be used to model certain aspects of relapse to

drug use, as in extinction/reinstatement procedures; however, this aspect is more often

investigated using self-administration paradigms (Myers et al., 2012; Tzschentke, 2007).

23

A more direct procedure to evaluate the reinforcing properties of a drug is to test

whether animals will work (usually lever press) to obtain the drug. This model

traditionally entails training an animal to self-administer a drug during daily sessions,

usually 1-3 hours in length. Most self-administration procedures use a fixed ratio (FR)

schedule of reinforcement. In this assessment, the animal must perform a fixed number

of responses to receive an intravenous drug infusion. If the animal finds the drug

rewarding, the number of responses that an animal will perform to obtain an infusion

generally increases. This is clearly seen in progressive ratio studies where the number of

responses required for the infusion is increased until the animal no longer responds

(Moser et al., 2011). Results from these kinds of studies have revealed that drugs can

serve as positive reinforcers and that there is correspondence between humans and

animals in terms of drugs that are self-administered and the pattern of drug intake. For

example, drugs that are abused by humans typically maintain responding in animals,

whereas drugs that do not maintain responding in animals are usually not abused by

humans (Collins et al., 1984). Similar patterns of drug intake have been reported in

humans and animals for opioids and other abused drugs (Griffiths, 1980).

1.3.3 Opioid Receptor Knockout Studies

Each gene encoding a receptor or peptide that is involved in the endogenous

opioid system has been knocked out in mice either singly or in combination (Gaveriaux-

Ruff and Kieffer, 2002).

The generation of a MOR knockout (KO) mouse confirmed the importance of the

MOR in mediating the analgesic and rewarding properties of opioid drugs. These mice

appeared normal morphologically, did not differ in weight when compared to wild-type

littermates, bred normally, and showed no changes in maternal behavior (Matthes et al.,

1996). However, MOR KO pups exhibited less attachment behavior when separated

from their mothers and were less responsive to their own mother’s cues (Moles et al.,

2004). The loss of MOR expression did not appear to significantly change other

components of the endogenous opioid system, nor were other components compensating

24

for this loss (Matthes et al., 1996; Sora et al., 1997). Tail flick, tail immersion, and hot

plate tests were used to confirm that MOR KO mice were insensitive to the analgesic

effects of morphine and other agonists (Kitanaka et al., 1998; Matthes et al., 1996; Sora

et al., 1997). Other responses to opioid drugs including respiratory depression and

slowed intestinal transit were also abolished in the absence of MORs (Dahan et al., 2001;

Matthes et al., 1998; Roy et al., 1998; Roy et al., 2001). Importantly, MOR KO mice

were insensitive to the rewarding effects of morphine and heroin in conditioned place

preference tests (Contarino et al., 2002; Matthes et al., 1996; Sora et al., 2001). A few

studies also found that self-administration of morphine and withdrawal precipitated by

naloxone was also abolished in these mice (Becker et al., 2000; Matthes et al., 1996; Sora

et al., 2001).

MOR KO mice have also provided a great deal of insight into the rewarding

properties of other drugs of abuse. Conditioned place preference for the active ingredient

of marijuana (Δ9-tetrahydrocannabinol) and nicotine were reduced when MORs were not

expressed (Berrendero et al., 2002; Ghozland et al., 2002). The self-administration of

cocaine and ethanol were also abolished in MOR KO mice (Mathon et al., 2005; Roberts

et al., 2000). Taken together, these data implicate the MOR in processing the rewarding

effects of other substances, not just opioid drugs. This also indicates that the endogenous

opioid system is involved in the regulation of reward processes.

Delta opioid receptor (DOR) knockout mice studies revealed that these mice

displayed higher levels of anxiety than MOR or kappa opioid receptor (KOR) knockouts

(Filliol et al., 2000). In a study by Roberts (2001), DOR KO mice were exposed to

alcohol in a two-bottle choice test. Naïve mutant mice and wild-type mice were

indistinguishable in their alcohol consumption. However, when tested in an operant

paradigm, the KO mice self-administered more alcohol than the wild-type mice.

Additionally, the elevated anxiety-like behavior in the DOR KO mice returned to wild-

type levels after alcohol administration. Therefore, the DOR may be an important factor

to explain the relationship between emotional states and the initiation and maintenance of

drug abuse. Interestingly, DOR-deficient mice do not develop tolerance to morphine,

25

which indicates that it may be involved in some kind of adaptive response to chronic

morphine exposure (Zhu et al., 1999).

Kappa and mu opioid receptors have largely been shown to oppose each other’s

effects. The KOR is involved in pain control, particularly visceral pain. KOR-deficient

mice showed no obvious change in the perception of mechanical or thermal pain;

however, they did show an enhanced response to peritoneal acetic acid injections. The

administration of kappa agonists typically leads to dysphoria. When KOR KO mice were

exposed to the kappa agonist, U 50,488H, conditioned place aversion was almost absent.

These mutant mice also displayed an attenuation of naloxone-precipitated withdrawal

syndrome when chronically treated with morphine (Simonin et al., 1998). This suggests

that the KOR participates in adaptations to long-term exposure to opioid drugs.

All three subtypes of opioid receptors appear to be potentially involved in some

aspect of addiction. A summary of the opioid receptor knockout mice studies is

presented in Table 1.5.

26

Table 1.5: Summary of opioid receptor knockout mice studies

Summary of responses to drugs and spontaneous behaviors observed in opioid receptor

knockout mice. Adapted from Gaveriaux-Ruff and Kieffer, 2002.

Knockout Drug Response Behavior in absence of drugs

MOR Morphine Decreased analgesia,

reward and dependence

Decreased locomotion

Increased thermal nociception

Decreased anxiety and depression

δ agonists Decreased analgesia,

reward and dependence

THC, alcohol Decreased reward

DOR δ agonists Decreased analgesia Increased locomotion

Increased anxiety, and depression

Morphine Decreased tolerance

Alcohol Increased intake

THC No change in reward

KOR κ agonists Decreased analgesia and

dysphoria

No change in locomotion

Increased visceral nociception

No change in anxiety or depression

Morphine Decreased Dependence

THC Decreased dysphoria

1.3.4 Physiological and Psychological Adaptations to Opioid Exposure

Our current understanding of opioid pharmacology suggests that activation of the

MOR by agonists, either endogenous or exogenous, is the most effective way to modulate

pain signals. However, DOR- and KOR-specific ligands have also demonstrated pain

modulation, but not to the same degree as MOR agonists.

27

How these drugs cause the development of tolerance, physical dependence, and

addiction is still poorly understood. Tolerance is the physiological process of adaptation,

where increasing amounts of the same drug are needed to achieve the same therapeutic

benefit. Physical dependence refers to the adaptive process whereby cessation of drug

use causes the onset of unpleasant withdrawal symptoms. Physical dependence can be

tested by treating individuals with an opioid antagonist like naloxone. One of the severe

side effects of opioid drugs is the development of addiction or substance dependence.

Addiction can be summarized as the inappropriate use of a drug despite negative

consequences (i.e. loss of job, criminal activity, poor health). The concern over

“creating” addicts has been shown to influence the prescribing habits of physicians,

leading to under-treatment of severe pain. For this reason, much effort has been focused

on a better understanding of these side effects and attempting to synthesize opioid

agonists that provide analgesia, but do not lead to tolerance, physical dependence, or

addiction. These efforts have resulted in an extensive library of agonists, antagonists, and

mixed agonists-antagonists; however, no compound has been developed that relieves pain

with no side effects.

Prolonged exposure to opioid agonists has been shown to cause pronounced

changes in the structure of neurons and the synaptic organization of the brain (Robinson

and Kolb, 2004). These changes are long lasting and may contribute to the characteristics

of addiction, including drug craving, compulsive drug use, and analgesic tolerance. For

example, in the ventral tegmental area chronic morphine exposure via subcutaneous

morphine pellets caused reductions in the overall size of the neurons in the mesolimbic

dopaminergic reward pathway. This long-term exposure also caused less branching and

fewer spines on the dendrites of medium spiny neurons in the shell of the nucleus

accumbens and on pyramidal cells in the prefrontal and parietal cortex (Robinson and

Kolb, 1999). The significance of these morphological changes is based on the

understanding that memory, learning, and other behavioral functions are associated with

the reorganization of synaptic connections in relevant neural circuits (Lamprecht and

LeDoux, 2004). Therefore, exposure to opioid drugs may be altering or creating neural

connections that potentiate drug-seeking behaviors. However, the susceptibility for

28

addiction is highly variable in individuals. Twin, family, and adoption studies have

shown that addiction is a complex genetic disorder that can be influenced by

environmental factors, such as home environment, parental support, socioeconomic

status, and education level (Lachman, 2006). This variable susceptibility has also been

documented in rats. Certain strains, such as Lewis, are more susceptible to drugs of

abuse than other strains, such as Fischer 344 (Suzuki et al., 1988). In Lewis rats,

morphine self-administration caused decreases in the branching and size of dendrites of

pyramidal cells in the motor cortex (Ballesteros-Yanez et al., 2007), but not in the Fischer

344 rats (Ballesteros-Yanez et al., 2008). When these two strains were compared prior to

morphine administration, Fischer 344 rats had neurons with shorter dendrites containing

fewer spines and branches when compared to neurons in Lewis rats (Ballesteros-Yanez et

al., 2008). These studies imply that significant changes to the structure of dendrites in

response to long-term opioid agonist exposure may contribute to susceptibility to

addiction.

Apart from its general inhibitory effects on dendritic structure, morphine has also

been shown to decrease neurogenesis in the hippocampus of adult rats (Eisch et al., 2000)

and to inhibit long-term potentiation (LTP) of excitatory synapses on CA1 neurons in the

hippocampus (Williams et al., 2001). Additionally, morphine has been shown to inhibit

the release of γ-aminobutyric acid (GABA) from neurons in the VTA (Nugent et al.,

2007). The exact role of neurogenesis in the hippocampus is not definitively known, but

recent evidence suggest that it plays an important role in integrating memories of events

that occur closely in time (Deng et al., 2010). Therefore, the inhibition of neurogenesis

by chronic morphine treatment may have deleterious effects on the function of this brain

region. The role of the MOR in mediating this effect was confirmed in a separate study

where it was found that the numbers and maturation of neurons in the granule cell layer

was increased in MOR knockout mice (Harburg et al., 2007). LTP is important for

synaptic plasticity, which has been implicated as a potential cellular mechanism that the

brain uses to encode activity- or experience-dependent events, such as learning and

memory (Cooke and Bliss, 2006). Presently, there is no clear relationship between LTP

and neurogenesis. Inhibition of LTP in the hippocampus and VTA may lead to abnormal

29

patterns of neural activity with unknown behavioral consequences. Thus, mechanisms

that are responsible for the structural and synaptic plasticity of the brain may play

important roles in the development of addiction. However, the specific mechanisms

linking opioid agonist activity to neuronal alterations observed with chronic opioid

exposure remain unknown.

1.3.5 Regulation of MOR

As GPCRs, MORs are regulated by the same mechanisms that regulate these

receptors, including endocytosis, recycling, and degradation (Drake et al., 2006;

Marchese et al., 2008). These regulatory mechanisms are usually initiated in response to

agonist receptor activation (Gainetdinov et al., 2004). Activated MORs undergo

internalization (or endocytosis) in response to most agonist drugs in most experimental

systems (Johnson et al., 2005). In general, agonist binding to MOR leads to downstream

signaling events mediated by Gαi/Gαo proteins. This now activated receptor can serve as

a target of G protein-coupled receptor kinases, which phosphorylate receptors at specific

threonine and serine residues. Phosphorylated receptors are then capable of binding to

arrestins that bind to clathrin and AP2, the clathrin-adaptor protein, which promotes

internalization of receptors by clathrin-coated pits. This process is known as homologous

desensitization (Gainetdinov et al., 2004). Following internalization, MORs are recycled

back to the surface during resensitization, while other opioid receptors, such as DORs,

are targeted to lysosomes for degradation and down-regulation (Tanowitz and von

Zastrow, 2003). Using chimeric MORs and DORs, a specific sequence present in the C-

terminal tail of the MOR was found to be both necessary and sufficient for recycling as

opposed to degradation. More recent studies have localized this sequence to the last 17

residues in the C-terminal tail called the MOR regulatory sequence (MRS) (Hislop et al.,

2009). The MRS promotes MOR recycling after short-term agonist exposure. The

essential trafficking of the MOR is controlled by the MRS, as mutant receptors lacking

this sequence are rapidly down-regulated. Some 3’ splice variants of the MOR lack the

MRS, which may account for increased tolerance to drugs that normally cause recycling

30

of the receptor (Pasternak et al., 2004). However, the MOR also undergoes agonist-

stimulated ubiquitination that promotes the sorting of receptors from the endosome

limiting membrane to the lumenal compartment. This ubiquitination of the MOR

functions independently and downstream of the ‘recycling versus degradation’ decision

of the MRS. More importantly, the ubiquitin-directed step is not required to direct

internalized MORs to lysosomes or to initiate their proteolysis, but does play a role in the

destruction of the MOR’s opioid binding site (Hislop et al., 2011). However, the

prototypical opioid agonist, morphine, does not cause endocytosis of MORs in human

embryonic kidney (HEK) 293 cells that have been stably transfected with the receptor

(Keith et al., 1996). Interestingly, the efficacy of morphine to produce internalization is

highly dependent on the system and brain region being examined (Johnson et al., 2005).

Brain slices from transgenic mice expressing epitope-tagged MORs in neurons of the

locus ceruleus showed no morphine-induced internalization of MORs (Arttamangkul et

al., 2008) and neither did MORs heterologously expressed in neurons of the hippocampus

(Bushell et al., 2002). In contrast, primary cultures of dissociated rat striatal neurons

showed rapid endocytosis of MORs in response to morphine (Haberstock-Debic et al.,

2005). These studies suggest that the expression of cellular components that regulate

MORs, such as GRKs and arrestins, are likely cell-type specific. For example, GRK2 has

been shown to phosphorylate MORs in HEK293 cell culture models (Schulz et al., 2004),

but has not been clearly shown in vivo (Johnson et al., 2005). However, a recent study on

the effect of beta arrestin on MOR regulation has shed some light on the reason for

morphine’s poor internalization. Mouse embryonic fibroblasts that were deficient in beta

arrestin 1, beta arrestin 2 or both were assessed for their ability to regulate MOR when

treated with morphine or DAMGO. DAMGO was shown to recruit both beta arrestin 1

and beta arrestin 2 to the MOR and either was sufficient to promote internalization.

DAMGO was also shown to promote ubiquitination of the MOR, which did not occur in

the absence of beta arrestin 1. In contrast morphine recruited beta arrestin 2 to the MOR,

but not beta arrestin 1. This interaction was sufficient to promote internalization of the

MOR, but not ubiquitination of the MOR. Taken together these results imply that beta

arrestin 1 is important in the ubiquitination of the MOR (Groer et al., 2011).

31

Despite these agonist-specific differences on MOR internalization, all agonists

produce similar physiological responses short-term (euphoria and analgesia) and

adaptation (tolerance and physical dependence) long-term. A seminal study from the lab

of Marshall Nirenberg attempted to correlate the development of tolerance and physical

dependence in animals with the specific effects that opioid agonists produce in cells.

This study showed that repeated administration of an opioid agonist on neurons

diminished their responsiveness to the drug as measured by the progressive dampening of

agonist-mediated cyclic adenosine monophosphate (cAMP) inhibition. When the agonist

was removed, the levels of cAMP rebounded above baseline (Sharma et al., 1975). This

suggested that receptor desensitization and subsequent cellular compensatory

mechanisms may underlie the development of tolerance and physical dependence.

Although numerous studies have continued to explore the mechanisms of MOR

regulation in an attempt to better understand the cellular context of tolerance, physical

dependence, and addiction; no clear mechanistic explanation for these adaptations has

been found (Bailey and Connor, 2005).

1.4 GPCR Interacting Proteins

The GPCR family is composed of a large and multifaceted group of cell surface

proteins that transduce extracellular signals into intracellular responses (Hill, 2006).

GPCR signaling is mediated by the activation of heterotrimeric guanosine triphosphate

(GTP)-binding proteins (G proteins), which act as secondary messengers to activate or

inhibit other proteins creating a cascade of events that produce a specific cellular

response (Hamm and Gilchrist, 1996). The versatility of these receptors is illustrated by

the varied physiological events they mediate, including sensory recognition (vision, taste,

and smell), endocrine regulation, and complex behavior phenomena (Gainetdinov et al.,

2004). In the central nervous system, these receptors serve as important targets for a

number of neuromodulatory drugs, such as psychotics and opioid analgesics (Hill, 2006).

Mounting evidence indicates that GPCRs bind to other proteins to form multiprotein

signaling complexes or signalplexes (Kabbani and Levenson, 2007; Milligan, 2005). The

32

interacting proteins have the potential to mediate previously unknown downstream

effects or to alter known mechanisms of receptor signaling and regulation (Bockaert et

al., 2010). For example, neuronal calcium sensor-1 (NCS-1) was shown to interact with

the D2 dopamine receptor (D2R) and interfere with its phosphorylation, which prevented

its endocytosis and desensitization (Kabbani et al., 2002). Since the MOR is known to

mediate the analgesic and rewarding properties of opioid drugs, proteins that interact with

the MOR likely play important roles in the development of tolerance, physical

dependence, and addiction to opioid drugs (Milligan, 2005). Identifying and

characterizing these proteins may provide insight into the mechanisms that underlie these

phenomena.

1.4.1 MOR Interacting Proteins

A comprehensive list of proteins that have been shown to interact with the MOR

is shown in Table 1.6. Many of these proteins affect the trafficking of the MOR by

promoting endocytosis (synaptophysin, PLD2, and M6a), helping to recycle and sort

internalized receptors (filamin A and GASP), or interfering with internalization

(RanBP9). Others affect the signaling properties of the MOR by inhibiting activation of

G-proteins (CaM and periplakin) or by inhibiting the ability of the MOR to inhibit cAMP

levels (PCK1) (Georgoussi et al., 2012). A novel signaling outcome for the MOR was

found when it was discovered that the MOR interacts with STAT5A. STAT5A is a

transcription factor and a member of the Signal Transducers and Activators of

Transcription family. When the MOR was activated, downstream transcriptional

activation of STAT-responsive reporter genes was detected in this study (Mazarakou and

Georgoussi, 2005). Although the role of these MORIPs has not been extensively studied

in terms of addiction, the identity of these proteins has contributed to the overall

knowledge of the MOR life cycle and downstream signaling events.

33

Table 1.6: Previously identified MORIPs

The location of the interaction (if known), a brief summary of the function of the

interaction and the original reference that identified the interaction are listed.

Interacting Protein Location

on MOR

Function Reference

Alpha 2A adrenergic

receptor

ND Receptor signaling (Jordan et al.,

2003)

Beta-arrestin 1

Beta-arrestin 2

ND Receptor desensitization and

endocytosis

(Bohn et al., 2000)

(Molinari et al.,

2010)

Calmodulin (CaM) IL3 Interferes with activation of G

proteins

(Wang et al., 1999)

Cannabinoid receptor

(CB-1)

ND Inhibition of signaling (Rios et al., 2006)

Delta opioid receptor

(DOR)

ND Modulation of signaling and ligand

binding

(Gomes et al.,

2000)

Filamin A C-tail Regulation of receptor recycling

and degradation

(Onoprishvili et

al., 2003)

Gai2/Gai3 IL3, C-tail Receptor signaling

(Georgoussi et al.,

1997)

Glycoprotein M6a ND Endocytosis and recycling of

receptor

(Wu et al., 2007)

G protein-regulated

inducer of neurite

outgrowth

IL-3 Regulates receptor distribution in

lipid rafts

(Ge et al., 2009)

Heat shock protein

HLJ1

C-tail ND (Ancevska-Taneva

et al., 2006)

Kappa opioid receptor ND Sex-dependent antinociception (Chakrabarti et al.,

2010)

Opioid receptor-like 1

receptor (ORL1)

C-tail Inhibits receptor signaling (Wang et al., 2005)

34

Periplakin C-tail Interferes with activation of G

proteins

(Feng et al., 2003)

Phospholipase D2

(PLD2)

C-tail Regulates receptor endocytosis (Koch et al., 2003)

Protein kinase Ci

(PKCi)

C-tail Negatively regulates receptor

desensitization

(Guang et al.,

2004)

Ran binding protein 9

(RanBP9)

C-tail Negatively regulates receptor

internalization

(Talbot et al.,

2009)

Regulator of G-protein

signaling 4 (RGS4)

C-tail Signaling and scaffolding (Georgoussi et al.,

2006)

RGS9-2 ND Receptor signaling and endocytosis (Garzon et al.,

2005a; Garzon et

al., 2005b)

Somatostatin receptor ND Internalization and desensitization (Pfeiffer et al.,

2002)

Spinophilin ND Modulates receptor signaling and

endocytosis

(Charlton et al.,

2008)

STAT5A C-tail Regulates receptor mediated

transcription

(Mazarakou and

Georgoussi, 2005)

Substance P receptor

(NK-1)

ND Receptor internalization and

resensitization

(Pfeiffer et al.,

2003)

Synaptophysin IL3 Modulates receptor signaling and

endocytosis

(Liang et al., 2007)

Wntless (WLS), GPR

177

IL2 Wnt secretion (Jin et al., 2010)

Of particular interest is the discovery of an interaction between MORs and

spinophilin, a scaffolding protein enriched in dendritic spines. Overexpression of

spinophilin in rat pheochromocytoma 12 (PC12) cells allowed the morphine-induced

endocytosis of the MOR within 30 minutes, an effect that was not seen in control cells.

In agreement with this finding, the loss of spinophilin expression in spinophilin KO mice

35

was found to increase measures of MOR signaling by delaying receptor internalization.

More importantly, in behavioral studies, spinophilin KO mice exhibited greater

sensitivity to the rewarding properties of morphine and developed a higher degree of

physical dependence (Charlton et al., 2008). This was the first study to show that the

behavioral response of an animal to opioid drugs could be affected by altering the

expression of a MOR interacting protein (MORIP). As more interactions and

multiprotein complexes are identified, researchers should be able to propose functional

roles for new associations, identify novel therapeutic targets, and devise more effective

strategies for encountering pathologies where the functionality of MORs is relevant

(Georgoussi et al., 2012). Thus, the continued identification and characterization of

MORIPs will be necessary to get a better understanding of the variety of functions that

are mediated by the MOR and the roles that these proteins may play in the pathogenesis

and treatment of complex neuropsychiatric diseases (Bockaert et al., 2010).

1.4.2 Identifying Candidate MORIPs

Many well-characterized methods exist to identify potential GPCR interacting

proteins. The conventional yeast two-hybrid approach was first introduced by Fields and

Song (1989) as a method for identifying an interaction between two proteins. This

technique relies on the reconstitution of the GAL4 yeast protein (a transcription factor),

which contains two distinct and separable functional domains: a DNA-binding domain

(BD) and a transcription-activating domain (AD). To test for an interaction, one protein

is fused with the BD and another is fused to the AD. Plasmid constructs are then

transformed into yeast cells that have been modified to have certain nutritional

requirements. If the proteins form an interaction, the binding and activating domains will

come into close proximity to one another and reconstitute the functional activity of the

GAL4 protein. This protein can then activate transcription and translation of the yeast

lacZ gene, which encodes for the enzyme β-galactosidase. This enzyme catalyzes the

chemical conversion of 5-bromo-4-chloro-3-indolyl-β-galactopyranoside to galactose and

5-bromo-4-chloro-3-hydroxyindole, which is visualized as a blue pigment when oxidized

36

to 5,5’-dibromo-4,4-dichloro-indigo. This method has been utilized as a screening

platform by the development of various cDNA libraries fused to the AD of the GAL4

protein. The selected library can then be tested against any ‘bait’ protein fused to the BD

of GAL4 (Miller and Stagljar, 2004). This approach is limited because the protein

interactions must take place within the nucleus of yeast cells. This technical issue makes

it difficult to use this conventional method with highly hydrophobic or membrane-bound

proteins (Lentze and Auerbach, 2008). In order to overcome these limitations, a

membrane-based yeast two-hybrid method was developed to enable the ability to detect

interactions between proteins that occur at the plasma membrane (Iyer et al., 2005;

Lentze and Auerbach, 2008). This method utilizes a split-ubiquitin molecule where the

N-terminal half (fused to a transcription factor) is fused to one protein and the C-terminal

half is fused to another protein. If an interaction occurs between the two proteins, the

ubiquitin molecule will be reconstituted and lead to the cleavage of the transcription

factor that is fused to the N-terminal half of the split-ubiquitin molecule.

1.5 Rationale and Hypothesis

Although opioid analgesics are efficacious for the treatment of pain, their use is

limited due to the side effects that they produce. The most undesirable side effect that

these drugs produce is the development of addiction. Unfortunately, the precise cellular

and molecular mechanisms that cause this effect are not yet known. What is known is

that these drugs activate the MOR and knockout studies have implicated the MOR as the

single opioid receptor subtype that mediates both the analgesic and rewarding properties

of these drugs. A growing body of evidence suggests that MORs are part of large

signaling complexes containing various MOR interacting proteins. Previously identified

MORIPs have been found to alter the regulation and signaling properties of the MOR.

Therefore, we hypothesize that identifying and characterizing novel MORIPs will help us

to better understand the cellular and molecular mechanisms that underlie the addiction

process.

37

In the following chapters, we examine the identification and validation of

interactions between the MOR and several novel MORIPs (Chapter 2), the functional

significance of the interaction between the MOR and novel MORIPs, seven in absentia

homologs 1 and 2 (Chapter 3), and the significance of the interactions between the MOR

and several novel MORIPs with opioid drug exposure (Chapter 4). The final chapter

discusses the potential relevance of the MOR-MORIP interactions in relation to MOR

regulation, possible implications for addiction, and directions for future research.

38

Chapter 2

Identification of MOR Interacting Proteins

2.1 Introduction

Most clinically relevant opioid agonists provide analgesia by activating MORs

(Berrettini et al., 1994; Inturrisi, 2002). MOR activation produces a euphoric effect in

many individuals, which is considered the primary motivational reward for abusing

opioid drugs (Bailey and Connor, 2005). The importance of the MOR in analgesia and

opioid dependence has been demonstrated by MOR knockout mice which show

significantly reduced sensitivity to both the analgesic and rewarding properties of opioids

(Contarino et al., 2002; Matthes et al., 1996). Like most GPCRs, the MOR is regulated

by multiple mechanisms, such as receptor desensitization, internalization, degradation,

and recycling (Petruzzi et al., 1997; Raehal and Bohn, 2005). The desensitization and

trafficking of MORs has been shown by a number of studies to represent key aspects in

the development of opioid tolerance and dependence (Bailey and Connor, 2005; Corbett

et al., 2006; von Zastrow et al., 2003; Waldhoer et al., 2004). Therefore, understanding

the mechanisms that regulate MOR signaling and trafficking is crucial for determining

the physiological basis of opioid dependence and enhancing opioid receptor

pharmacology for the treatment of addiction and pain.

A number of studies have shown that MOR desensitization and receptor

trafficking can increase the rewarding properties of opioid drugs, while reducing the

development of opioid tolerance and addiction-like behaviors (Berger and Whistler,

39

2011; Finn and Whistler, 2001; He et al., 2002; He et al., 2009; Martini and Whistler,

2007; von Zastrow et al., 2003; Whistler et al., 1999). However, the specific molecular

mechanisms that regulate these processes are largely unknown. Elucidating the

mechanisms that regulate MOR signaling and trafficking is critical for determining the

cellular response to opioid agonist drugs and for opening new avenues of investigation

into the pharmacotherapy of pain management.

Evidence indicates that GPCR signaling is modulated by proteins that bind to

form mulitcomponent protein assemblies, or signalplexes, involving interactions between

themselves, other GPCRs, and with other interacting proteins (Kabbani and Levenson,

2007; Milligan, 2005). A number of proteins have been identified and shown to affect

MOR biogenesis, trafficking, and signaling. For example, β-arrestin, calmodulin, and

synaptophysin have all been found to bind to the third intracellular loop of the MOR

(Georgoussi et al., 2011). Arrestins have been shown to be key components of MOR

desensitization because they can block the receptor’s interaction with G-proteins and can

promote recycling of the receptor through interactions with the clathrin assembly

machinery and clathrin-dependent endocytosis (Bohn et al., 2000; Molinari et al., 2010).

Calmodulin is believed to compete with G-protein for binding to the receptor and may

serve as an independent second messenger molecule released in response to MOR

stimulation (Wang et al., 1999). Synaptophysin is a major integral membrane

glycoprotein found in synaptic vesicles that interacts with a variety of nerve terminal

proteins such as dynamin I and adaptor protein 1 (AP-1). It is believed that

synaptophysin recruits dynamin to the plasma membrane to facilitate fusion of clathrin-

coated vesicles so that MOR can be endocytosed (Liang et al. 2007). The complete list of

40

currently known MOR interacting proteins along with a short description of the

functional significance of each interactor is shown in Table 1.6 of Chapter 1.

To better understand the potential role of MORIPs in the MOR life cycle, we have

performed conventional and modified yeast two-hybrid (Y2H) studies designed to

identify novel constituents of the MOR signalplex. Previous interaction screens for

MORIPs have primarily utilized the third intracellular loop (IL3) or the C-terminal tail

(Ctail) of the MOR as bait (Georgoussi et al., 2012). Previous studies in our laboratory

identified several D2 dopamine receptor interacting proteins, using the second

intracellular loop as bait in a conventional yeast two-hybrid screen (Kabbani and

Levenson, 2007). Therefore, we utilized the second intracellular loop as well as the

entire MOR to screen human brain cDNA libraries in order to expand the growing list of

MORIPs. Using these approaches, we have identified ten novel MOR binding partners.

Functional characterization of these interacting proteins will serve to further our

understanding of the mechanisms regulating MOR-mediated signaling and may help

elucidate the underlying molecular basis of cellular response to opioid agonist drugs.

2.2 Experimental Procedures

2.2.1 Membrane Yeast Two-Hybrid Screen

A modified split-ubiquitin yeast two-hybrid (MYTH) screen was performed in

collaboration with Igor Stagljar (University of Toronto) as described previously (Iyer et

al., 2005; Kittanakom et al., 2009; Stagljar et al., 1998). Briefly, the full-length human

41

MOR (transcript MOR-1) cDNA was cloned into the bait vector pCCW-STE

(Dualsystems Biotech AG, Switzerland) and the human fetal brain cDNA library was

cloned into the prey vector pRP3-N (Dualsystems). These constructs were then

sequentially transformed into S. cerevisiae reporter strain THY.AP4. This transformation

yielded 6 x 106 transformants/μg DNA on synthetic dropout (SD) agar plates (-Trp/-Leu/-

His/-Ade; Clontech, Palo Alto, CA) containing 3-amino-1,2,4-triazole (3AT). The

identity of the putative MORIPs was determined by recovering the prey plasmid from the

transformed yeast colonies. The cDNA clone in the plasmid was sequenced using

primers designed against the regions flanking the cloning site. These sequences were

then analyzed using Basic Local Alignment Search Tool (BLAST) to determine the

identity of the clone.

2.2.2 Conventional Yeast Two-Hybrid Screen

Two conventional yeast two-hybrid screens were performed as described

previously (Lin et al., 2001; 2005) using either the second intracellular loop (IL2; amino

acids 166-187) or the C-terminal tail (C-tail; residues 361-420) of the MOR as bait to

screen a fetal human brain cDNA library (Clontech). The MORIL2 and MOR C-tail

were cloned into the yeast GAL4 DNA binding domain expression vector pAS2-1

(Clonetech), while the human fetal brain cDNA library was provided in the GAL4

activation domain vector pACT2 (Clontech). Bait and prey plasmids were successively

transformed into yeast strain MaV103. Transformation of the yeast with MORIL2 and

the fetal human brain library produced approximately 2 x 106

transformants/μg DNA on

42

quadruple dropout plates (-Leu/-Trp/-His/-Ura; Clontech) containing 3AT.

Transformation of the yeast with MOR C-tail and the fetal human brain library yielded no

colonies on transformation efficiency plates; therefore the number of transformants/μg

DNA could not be determined. Interactions were assayed for β-galactosidase (β-gal)

activity via the nitrocellulose lift method (Lin R, 2005). cDNAs were extracted from

yeast colonies, sequenced, and subjected to BLAST analysis to determine their identities.

2.2.3 Mapping of Binding Regions

To map sites of interaction between the MOR and the newly identified MORIPs,

each MOR intracellular loop (IL) was tested for interaction with individual MORIPs

using the traditional Y2H method. MOR IL domains (IL1, amino acids 97-102; IL2,

amino acids 166-187; IL3, amino acids 259-282; and C- tail residues 361-420) were

separately ligated into pAS2-1 (Clontech) and assayed for interaction with candidate

MORIP cDNA clones in pACT2. Bait and prey plasmids were simultaneously co-

transformed into S. cerevisiae strain MaV103 and interactions were assayed for β-gal

activity as described above.

2.2.4 Glutathione S-transferase Pulldown Assay

MORIP-GST (glutathione S-transferase) fusion proteins were constructed by

separately fusing cDNAs encoding human AUP1 (residues 1-410), human DOK4

(residues 1-326), human CSN5 (residues 1-335), or the second intracellular loop of the

43

mu-opioid receptor (MOR-IL2; residues 166-187) to GST in the expression vector

pGEX-4T-1 (Amersham Biosciences, Piscataway, NJ). The polymerase chain reaction

(PCR) primers used for this cloning can be found in Table 2.1. cDNAs encoding the

MOR-IL2 (residues 166-187), SIAH1 (residues 1-233) and RanBP9 (residues 170-381)

were subcloned into the pET30a expression vector (Novagen, Madison, WI) containing

an S-tag. Fusion proteins were induced in Escherichia coli strain BL21 (DE3) using the

ZYP-5052 auto-induction media as described previously (De Cotiis et al., 2008; Studier,

2005). MORIP-GST fusion proteins bound to glutathione sepharose beads (GE

Healthcare, Piscataway, NJ) were used to pull down S-tagged proteins from bacterial

lysates as previously described (Jin et al., 2010; Lin et al., 2001). Eluted proteins were

separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) filter for

western blot analysis. The filter was probed with a horseradish peroxidase (HRP)-

conjugated anti-S-tag antibody (1:5000 dilution, Novagen, Madison, WI), and

immunoreactivity detected by enhanced chemiluminescence with an ECL Plus kit (GE

Healthcare).

44

Table 2.1: PCR primers used for cloning MORIPs

This table lists the PCR primers that were used to clone MORIPs into pET-30a to

produce S-tagged proteins or into pGEX-4T-1 to produce GST-fusion proteins for use in

GST pull down experiments.

Construct Direction PCR primer sequence

SIAH2 S-tag Forward GAT ATC ATC AGG AAC CTG GCT ATG GAG AAG GTG

Reverse CTC GAG GGC CAA CCC CAT TCA TCC CAG GT

SIAH1 S-tag Forward GAT ATC ATG AGC CGT CAG ACT GCT ACA GCA TTA CC

Reverse CTC GAG TCA ACA CAT GGC AAT AGT TAC ATT GAT GCC

CSN5-GST Forward GGA TCC GTA AAG TTG CGT CTT GGT TGT

Reverse GAA TTC TGT CTT TCA GGT AAA GTA CTT CTC AGA GAC TGT

DOK4-GST Forward GGA TCC ATG GCG ACC AAT TTC AGT GAC ATC GTC AAG

Reverse GAA TTC CTG TGG TCA CTG GGA TGG GGT CTT

AUP1-GST Forward GAA TTC ATG GAG CTT CCC TCA GGG CCG

Reverse CTC GAG GTT CCT TTG AGC TCA GTC AGC CTC

MORIL2 S-tag Forward GGG GAA TTC GAT CGA TAC ATT GCA GTC TGC CAC CCT

MORIL2 GST Reverse GGG CTC GAG TTT GGC ATT TCG GGG AGT ACG GAA ATC

RanBP9-GST Forward GGA TCC GCC GTG GAC GAA CAA GAG ACG

Reverse CTC GAG GGT CTG CCA TTC TCC TCG ATC

2.2.5 Cell Culture

Human embryonic kidney (HEK) 293 cells stably transfected with either FLAG-

tagged mu-opioid receptor (HEK-MOR), delta opioid receptor (HEK-DOR), or kappa

opioid receptor (HEK-KOR) were maintained in Dulbecco's Modified Eagle's Medium

45

(DMEM) supplemented with 10% fetal bovine serum (FBS) and 400 μg/mL G418.

HEK-MOR and HEK-DOR cell lines were generously provided by Dr. Mark van

Zastrow (University of California San Francisco). HEK-KOR cells were a gift from Dr.

Lee-Yuan Liu-Chen (Temple University School of Medicine).

2.2.6 Co-immunoprecipitation and Western blot Analysis

HEK-MOR, DOR, or KOR cells (stably expressing FLAG-tagged MOR, DOR, or

KOR, respectively) were separately transfected with constructs encoding full-length

MORIPs sub-cloned in the pCMV-Tag3B expression vector (Stratagene, La Jolla, CA)

containing a myc-tag. Transfections were carried out using Effectene transfection

reagent (Qiagen, Valencia, CA). Cells were cultured for 24 hours in DMEM and then

lysed in 1X lysis buffer (20 mM Tris-HCl pH7.5, 150 mM NaCl, 1 mM Na2EDTA, 1

mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4,

1% Triton X-100 and 1mg/mL leupeptin) supplemented with protease inhibitor cocktail

(Pierce, Rockford, IL). MOR, DOR, and KOR were immunoprecipitated from total cell

lysates using a polyclonal rabbit anti-FLAG antibody (Sigma, St. Louis, MO) coupled to

Protein-G Dynabeads (Invitrogen). Western blot analysis of immunoprecipitated

complexes was performed by using a monoclonal mouse anti-myc antibody (1:5000

dilution; Millipore, Billerica, MA) and a rabbit anti-mouse HRP-conjugated secondary

antibody (1:20,000 dilution; Jackson ImmunoResearch, West Grove, PA).

Immunoreactivity was detected by ECL Plus.

46

2.2.7 Immunofluorescence Microscopy

Immunohistochemistry was performed by transfecting HEK-MOR as described

for the co-immunoprecipitation. Cells were then split onto collagen coated coverslips

(BD Biosciences) and allowed to grow overnight. After 18 hours, cells were fixed with

4% paraformaldehyde and 5% sucrose. The cells were immunostained with monoclonal

mouse anti-myc antibodies (1:1000 dilution; Millipore) and rabbit anti-MOR antibodies

(1:500; AB5511, Millipore). After this initial staining, the cells were washed with 1X

PBS, and exposed to FITC conjugated donkey anti-rabbit antibodies (1:500 dilution;

Jackson ImmunoResearch) and rhodamine conjugated donkey anti-mouse antibodies

(1:500 dilution; Jackson ImmunoResearch). Images were obtained with a confocal

microscope (Zeiss LSM 510 Meta, Carl Zeiss Inc., Thornwood, NY), and digital images

were captured and imported with the LSM 5 image browser (Carl Zeiss, Inc.).

2.3 Results

2.3.1 Identification of Novel MOR Interacting Proteins

In the MYTH screen, 104 positive clones were obtained from the 6 x 106

colonies

screened. The putative MOR interacting proteins identified in this screen can be found in

Table 2.2. Sequence analysis identified GPR177 (WLS) (Jin et al., 2010), a putative

multi-pass membrane-spanning protein that is the mammalian ortholog of Drosphila

Wntless/Evi/Sprinter (reviewed in Hausmann et al., 2007). WLS plays an important role

in Wnt protein secretion, a process that is inhibited by the opioid agonist morphine (Jin et

47

al., 2010). Members of the Wnt pathway, such as WLS, are of particular interest because

they have been shown to affect neuronal development (Salinas and Zou, 2008). Many of

these Wnt-mediated effects mirror the effects induced by opioid agonist activity, but with

opposite outcomes. For example, opioid drugs have been shown to inhibit neurogenesis

in the adult rat hippocampus (Eisch et al., 2000), whereas Wnt proteins have been

demonstrated to promote neurogenesis in the adult rat hippocampus (Lie et al., 2005).

Vesicle-associated membrane protein-A (VAPA or VAP33) plays a role in endoplasmic

reticulum to Golgi transport (Peretti et al., 2008; Wyles et al., 2002). The number and

activity of opioid receptors at the cell surface are important in determining their capacity

to modulate downstream signaling. The receptor number at the cell surface can be

regulated through the biosynthesis pathway, including transcription, translation, protein

folding, and transport (Chen et al., 2006a; Chen et al., 2006b; Petaja-Repo et al., 2000).

Although the general consensus is that total opioid receptor protein does not change in

response to most opioid drugs (Christie, 2008; Johnson et al., 2005; Patel et al., 2002;

Stafford et al., 2001). This is not always the case. In a study using rat nucleus

accumbens, acute morphine treatment was shown to cause an increase in the amount of

epitope-tagged MORs in neuronal processes (Haberstock-Debic et al., 2003). Therefore,

proteins involved in the biosynthetic pathway may play an important role in responses to

opioid drugs. Connexin 37 (Cx37), and voltage-gated potassium channel Kv5.1 were

also identified in the MYTH screen (Table 2.1). These proteins play a role in, gap

junction formation (Haefliger et al., 1992), and regulation of resting membrane potential

(Kramer et al., 1998), respectively. Gap junctions mediate electrical transmission

between neurons by providing a low resistance pathway for the spread of electrical

48

currents and small metabolites (Bennett, 1997), while voltage-gated potassium channels

help to repolarize neurons after an action potential has been generated. Changes in

electrical transmission can contribute to synaptic plasticity in the form of long-term

potentiation (strengthening of synapses) or long-term depression (weakening of synapses)

(Dacher and Nugent, 2011). Thus, these two proteins may be important in the synaptic

plasticity observed with opioid addiction.

Table 2.2: Putative MORIPs identified in MYTH screen

ADD3 Adducing gamma subunit

GJA4 Gap junction protein, alpha 4

GPR177 Orphan g-protein coupled receptor

KCNF1 Potassium channel subunit

SLC31A2 Putative copper transporter

SLC39A13 Putative zinc transporter

SLC9A9 Putative Na+/H

+ exchanger

VAP33 VAMP-associated protein A

WHSC2 Wolf-Hirschhorn syndrome candidate 2

YIPF3 Yipl domain family, member 3

In the conventional yeast two-hybrid screen using the second intracellular loop

(IL2) of the MOR as bait to screen a fetal human brain cDNA library, 20 positive clones

were obtained from the 2 x 106 colonies screened. Sequence analysis identified several

clones encoding components involved in the ubiquitin pathway and in signal transduction

(Table 2.3). These included COP9 subunit 5 (CSN5), a protein proposed to regulate

49

exosomal protein sorting in both a deubiquitinating activity-dependent and -independent

manner (Liu et al., 2009), ancient ubiquitous protein 1 (AUP1), a protein that promotes

ubiquitination of misfolded proteins (Mueller et al., 2008), and seven in absentia

homologs 1 and 2 (SIAH1 and SIAH2), known really interesting new gene (RING) finger

E3 ubiquitin ligases (Hu et al., 1997a). The ubiquitin pathway has been shown to

mediate synaptic plasticity, learning and memory, and neurogenesis (Bingol and Sheng,

2011; Tuoc and Stoykova, 2010). Thus, these proteins were of interest for further study.

Additionally, four putative scaffolding proteins, docking protein 4 (DOK4), docking

protein 5 (DOK5), zyxin, and Ran binding protein 9 (RanBP9) were also identified as

candidate mu-opioid receptor interacting proteins (Table 2.3). Scaffolding proteins bind

and bring together two or more signaling proteins. In doing so, these proteins can direct

the flow of information by activating, coordinating, and regulating signaling events

(Buday and Tompa, 2010). Drugs of abuse can alter MOR signaling (Bailey and Connor,

2005; Christie, 2008), so these types of proteins are of interest for further study.

50

Table 2.3: Putative MORIPs identified in conventional yeast two-hybrid screens

MORIP NAME BAIT RESIDUES

AUP1 Ancient ubiquitous protein 1 IL2 321-410

CSN 5 Constitutive photomorphogenic signalosome 9

subunit 5

IL2 25-318

DOK4 Downstream of kinase 4 IL2 65-289

DOK5 Downstream of kinase 5 IL2 40-306

PLAGL1 Pleiomorphic adenoma gene-like 1 IL2 28-165

PLXNA4 Plexin A4 C-tail Non-coding

RanBP9 RAN binding protein 9 IL2, C-tail 88-366

SIAH1 Seven in absentia homolog 1 IL2 1-233

SIAH2 Seven in absentia homolog 2 IL2 122-324

Zyxin Zyxin IL2 203-483

In the conventional yeast two-hybrid screen using the C-terminal tail of the MOR

as bait to screen a fetal human brain cDNA library, 7 positive clones were obtained in the

screen. The transformation efficiency plates did not produce yeast colonies, so the

estimation of transformation efficiency could not be determined. Sequence analysis

identified RanBP9, which was identified in the screen using MORIL2, and plexin A4, a

protein that is involved with axonal guidance (Haklai-Topper et al., 2010) (Table 2.2).

However, the portion of plexin A4 pulled out in the screen was part of the non-coding

region, and was not pursued further.

51

2.3.2 Mapping of Binding Regions

The split-ubiquitin screen used the entire coding region of MOR as bait; therefore,

this approach provided no information regarding the physical location of MORIP binding

sites on the MOR. To map the binding site of the MORIPs on the MOR, the

conventional yeast two-hybrid system was used to interrogate interaction of individual

MORIPs with each of the MOR intracellular loops and the C-terminal cytoplasmic tail

(Ctail). Each of the MORIPs identified in the split-ubiquitin screen was found to interact

with only the second intracellular loop of the MOR (Figure 2.1). This result was

somewhat unexpected, as previously discovered MORIPs have been found to bind to IL3,

IL2, and the Ctail. Therefore, it is a little unsual that all of the MORIPs discovered in the

MYTH screen interacted only with the second intracellular loop. However, this data does

suggest that the second intracellular loop of the MOR is an important site for protein-

protein interactions.

The MORIPs identified in the screen using MORIL2 as bait were also tested,

because some GPCR interacting proteins have been shown to interact with more than one

intracellular region of GCPRs (Cen et al., 2001; Georgoussi et al., 2006). Each of the

MORIPs from the conventional screens interacted, as expected, with the second

intracellular loop (IL2) of the MOR (Figure 2.1). Two of the MORIPs, CSN5 and

RanBP9, also interacted with the first intracellular loop (IL1), while AUP1, CSN5,

DOK4 and RanBP9 were found to interact with the C-terminal tail of the receptor. This

is consistent with RanBP9 being pulled out in the screen using the MOR Ctail as bait.

The binding site for Cx37 was not tested in this assay. These results suggest that the

52

MORIPs identified interact with specific domains of the MOR in the yeast system, and

that the second intracellular loop of the receptor is an apparent hot-spot for protein-

protein interactions.

Figure 2.1: Mapping of MORIP binding regions

Portions of each MORIP were tested in a directed Y2H assay for interaction with each of

the intracellular loops (IL) and carboxyl-terminus (C-tail) of the MOR. Empty bait

vector (pACT2) was used as a negative control. A positive interaction is indicated by the

production of a blue colony in the β-galactosidase assay.

53

2.3.3 Validation of MOR/MORIP interaction by GST Pulldown

To further validate direct interaction between newly identified MORIPs and the

MOR, we tested the ability of selected MORIP-GST fusion proteins to associate with S-

tagged MORIL2 in a pull-down assay. Western blots containing lysates from bacteria

expressing an S-tagged MORIL2 cDNA produced an immunoreactive band of ~13 kDa

when probed with anti-S-tag antibodies (Figure 2.2). This band corresponds to the

expected size of the MORIL2 encoded by the cDNA construct. The same band was

detected by pull-down after the bacterial lysate was incubated with either the CSN5-GST,

AUP1-GST, or DOK4-GST fusion proteins, but not when the lysate was absorbed onto

beads alone or GST-coated beads. We also utilized the pull-down assay to test the ability

of RANBP9, SIAH1, and zyxin to associate with a MORIL2-GST fusion protein. As

shown in Fig. 2.2, S-tagged RANBP9 cDNA produced an immunoreactive band of ~34

kDa, S-tagged SIAH1 cDNA produced a band of ~32 kDa, and S-tagged Zyxin was

detected at ~85 kDa when probed with anti-S-tag antibody. These bands correspond in

size to the predicted sizes of the protein fragments encoded by the respective cDNA

constructs, and were not detected when the lysates were absorbed onto beads alone or

GST-coated beads.

54

Figure 2.2: GST pull-down of MORIPs and MOR

GST pull-down assays were performed to interrogate the interaction between selected

full-length MORIPs and the MORIL2 domain. In the top three panels, MORIP-GST

fusion proteins were used to pull down the S-tagged MORIL2. In the bottom three

panels, MORIL2-GST fusion proteins were used to pull down S-tagged MORIPs. Pull-

down products were purified on glutathione beads, separated by SDS-PAGE, and probed

on Western blots using HRP-conjugated anti-S-tag antibodies. S-tagged MORIPs or

MORIL2 domains produced in bacteria are shown in lysate lanes (Ly), while uncoated

glutathione sepharose beads (Beads) or GST-coated glutathione sepharose beads (GST)

incubated with S-tagged proteins served as negative controls. PD indicates pull-down

lanes.

55

2.3.4 Interaction of MORIPs with MOR, DOR, and KOR in Cultured Mammalian Cells

The interaction between full-length MOR and selected MORIPs was verified

using co-immunoprecipitation (co-IP) experiments. To demonstrate an interaction in cell

culture, the ability of an anti-FLAG antibody to co-IP MORIPs from lysates prepared

from HEK 293 cells stably expressing FLAG-tagged MORs (HEK-MOR) was tested.

Probing Western blots with anti-myc antibodies revealed immunoreactive bands of the

expected sizes in lysate lanes (L) prepared from HEK-MOR cells transiently transfected

with myc-tagged AUP1, CSN5, Cx37, DOK4, VAPA cDNAs (Figure 2.3B). Anti-

SIAH1, anti-SIAH2, and anti-WLS antibodies detected immunoreactive bands of the

expected sizes in lysate lanes for SIAH1, SIAH2, and WLS, respectively, in

untransfected cells. In co-IPs where no anti-FLAG antibody was added (mock, M), no

such bands were detected; however, bands migrating at similar molecular weights were

detected in the IP lanes. Y2H and co-IP experiments have recently confirmed an

interaction between RANBP9 and the MOR (Talbot et al., 2009). Taken together, these

results support the view that the MORIPs identified in our Y2H screens interact with full

length MOR in the context of cultured mammalian cells.

56

Figure 2.3: Co-immunoprecipitation of MORIPs and MOR, KOR, and DOR

(A) Amino acid sequence comparison between MOR, DOR, and KOR IL2 domains. (B)

Co-immunoprecipitation of full length MORIPs with MOR, DOR, or KOR. Flag-tagged

MOR, DOR, or KOR was immunoprecipitated from HEK-MOR, HEK-DOR, or HEK-

KOR cells, respectively, using rabbit anti-flag antibodies. Mock immunoprecipitations

were performed with Protein-G beads coated with non-specific rabbit IGG. Blots were

probed with either anti-MORIP antibodies for the presence of endogenously expressed

MORIPS (SIAH1, SIAH2 and WLS) or with anti-myc antibodies for transiently

transfected myc-tagged MORIPs. Lysate lanes (L) contain 5% of the total protein

compared to the mock (M) and immunoprecipitation (IP) lanes.

The Y2H and pull-down data indicate that each of the MORIPs identified in the

screens interacts with the second intracellular loop of the MOR. Sequence comparisons

indicate that the second intracellular loops of the MOR, DOR and KOR exhibit a high

57

degree of amino acid sequence homology (Figure 2.3A). Among the opioid receptor

proteins, the second intracellular loop exhibits 85-90% amino acid sequence similarity,

with the highest homology within the N-terminal portion of the loop (Figure 2.3A).

Because of this homology, we used co-IPs to determine whether any of the MORIPs also

interacted with other opioid receptor family members. To do this, we tested the ability

of anti-FLAG antibodies to co-IP MORIPs from lysates prepared from HEK-DOR and

HEK-KOR cells (stably expressing FLAG-tagged DORs and KORs, respectively). As

shown in Figure 2.3B, AUP1-myc, Cx37-myc, SIAH1, SIAH2, VAPA-myc, and WLS

were able to interact with both DOR and KOR. However, neither CSN5-myc nor DOK4-

myc showed interaction with DOR or KOR, despite the fact that the proteins were

expressed in the transfected cells, as indicated by the presence of immunoreactive bands

of the appropriate size in the DOR and KOR lysate lanes (Figure 2.3B). Together, these

results suggest that many of the MORIPs we identified are also capable of interaction

with the DOR and KOR, at least within the context of transfected mammalian cells. The

interaction of these proteins with each of the opioid receptor family members will depend

in large part on whether an opioid receptor family member and a particular opioid

receptor binding protein are co-expressed within the same cell (and cellular

compartment) within the nervous system.

2.3.5 Immunofluorescence Microscopy

Immunofluorescence microscopy was performed to determine the cellular

location of MORIPs and MOR in HEK-MOR cells. Due to the lack of available

58

antibodies to selected MORIPs or their use in immuohistochemistry, myc-tagged proteins

were transfected into HEK-MOR cells. Cells were double labeled with mouse anti-myc

and rabbit anti-FLAG antibodies, donkey anti-mouse FITC-conjugated, and donkey anti

rabbit rhodamine-conjugated secondary antibodies to label the MORIPs green and MORs

red. If the MOR and the MORIP signals overlap (orange or yellow), it suggests that the

proteins occupy the same space within the cell, and therefore are able to interact.

However, the observed staining of the MOR is not entirely at the membrane, as would be

expected (Figure 2.4). This may be due the overexpression of the MOR in these cells or

to non-specific staining by the rabbit anti-MOR antibodies. Therefore, conclusions cannot

be made in regards to the possible co-localization of the MOR with these MORIPs. The

immunofluorescence does allow the approximate intracellular location of the MORIPs to

be determined. DOK4 was observed primarily near the plasma membrane (Figure 2.4),

which is consistent with a previous report (Uchida et al., 2006). AUP1, SIAH1, and

CSN5 were all predominantly expressed in the cytosol (Figure 2.4). As with DOK4, the

cellular locations of AUP1, SIAH1, and CSN5 are consistent with previous reports (Hu

and Fearon, 1999; Kato et al., 2002; Luo et al., 2006).

59

Figure 2.4: Co-localization of MOR and MORIPs

MOR was labeled with rabbit anti-MOR antibody and donkey anti-rabbit FITC. Myc

fusion proteins were visualized using mouse anti-myc antibody and donkey anti-mouse-

rhodamine. Images are confocal. Overlay shows the merging of the labels and is

indicated by a yellow color.

2.4 Discussion

GPCR signaling is modulated by proteins that bind to form signalplexes,

involving interactions between themselves, other GPCRs, and with other interacting

proteins. As a GPCR, the MOR is expected to have an extensive signalplex (Kabbani

and Levenson, 2007; Milligan, 2005). Utilization of conventional and modified yeast

two-hybrid (Y2H) methods identified ten novel and two previously reported components

of this complex. Directed Y2H and GST-PD studies were used to confirm interaction

60

and map the binding site for several MORIPs to the second intracellular loop of MOR.

Many MORIPs were demonstrated to interact with full length MOR in the context of

cultured human cells. Due to high sequence homology in intracellular loop two, many

MORIPs also interact with the KOR and DOR, suggesting that these proteins may be

universal regulators of opioid function

Previous conventional Y2H screens have identified many of the known MOR

interactors including periplakin, protein kinase C I (PKCI), phospholipase D2 (PLD2),

synaptophysin, glycoprotein M6a, filamin A, and DnaJ-like 1 protein (Hlj) (Georgoussi et

al., 2012). The conventional Y2H presented here was unique in that it utilized the

second intracellular loop of MOR, a region of the receptor that was previously unstudied

in terms of interacting proteins. This screen yielded six novel interactors and two

previously reported MORIPs (wntless and Ran binding protein 9). In addition, a

modified split-ubiquitin Y2H screen identified four interactors of full length MOR. The

advantage of this type of screen is that full length membrane proteins can be used as bait,

and the screen is able to identify membrane-localized interactors which may be missed in

the conventional Y2H screen.

Although these screens were successful in discovering novel MORIPs, the Y2H

method can result in false negative and false positive results. False positive results are

usually eliminated during subsequent analysis, such as GST pull-down and co-IP. False

negative results can occur for a variety of reasons. For example, if the interaction

depends on posttranslational modifications, such as glycosylation or phosphorylation,

these modifications may not occur properly or at all in the yeast system. Therefore, these

interactions would not be detected in the Y2H. Since the fusion proteins must be targeted

61

to the nucleus, hydrophobic proteins (such as membrane bound proteins) or proteins with

strong targeting signals may not reach the nucleus to activate the reporter gene and would

also be missed in the Y2H. Additionally, very weak or transient interactions are not

usually detected using this method and would likely be missed in a screen. These

disadvantages in using the Y2H method are important, because they help explain why

more interacting proteins were not found.

These studies have shown via directed Y2H that several MORIPs are capable of

interacting with multiple loops of the MOR. This is not surprising as some previously

identified interactors of MOR and DOR have been shown to be able to associate with

more than region of the receptor. Regulators of G protein signaling 4 protein (RGS4) and

beta arrestin have been demonstrated to interact with both the third intracellular loop and

the C-terminal tail of the DOR (Cen et al., 2001; Georgoussi et al., 2006).

Heterotrimeric G proteins (Gi) bind to the MOR at the third intracellular loop and the C-

terminal tail (Georgoussi et al., 1997).

The MORIPs identified in this and other studies are functionally diverse, but

many share common functions including protein processing, cellular localization, signal

regulation, receptor desensitization, ubiquitination, scaffolding, WNT secretion, and axon

guidance. Several of the novel MORIPs identified in the current screens belong to the

ubiquitin proteasome pathway. Seven in absentia homolog 1 and 2 (SIAH 1 and 2) are

RING finger E3 ubiquitin ligases that mediate ubiquitination and subsequent proteasomal

degradation of target proteins (Hu et al., 1997a). COP9 subunit 5 (COP9S5 or CSN5) is

one subunit of the large COP9 signalosome (CSN) which is responsible for the

deneddylation and activation of Cullin ring E3 ubiquitin ligases that ubiquitinate targets

62

for degradation (Choo et al., 2011). These MORIP/MOR interactions are surprising

considering that MOR is typically downregulated by lysosomal degradation and not the

proteasome. However, ubiquitination of MOR does occur and is required for trafficking

events that downregulate receptor ligand binding (Hislop et al., 2011). Since the E3

ligase for ubiquitination of MOR is unknown, it will be important to determine whether

SIAH1, SIAH2, or CSN5 promotes ubiquitination of the opioid receptors. An alternative

hypothesis is that MOR can act to scaffold these proteins for ubiquitination of other

MORIPs. In fact, synaptophysin, a previously identified MORIP is a target of both

SIAH1 and SIAH2 (Wheeler et al., 2002). There is also mounting evidence that the

ubiquitin pathway is involved in a variety of neuronal activities, including dendritic

morphogenesis and synaptic plasticity (Bingol and Sheng, 2011; Tuoc and Stoykova,

2010; Yang et al., 2008). Similarly, chronic opioid exposure has been shown to lead to

changes in dendritic arborization and synaptic plasticity (Robinson and Kolb, 2004). To

date there is no direct evidence to connect these observations.

Two proteins from the downstream of kinase (DOK) family of adaptor proteins

were also identified as interactors of MORIL2. This family of proteins has a broad range

of functions. In the nervous system, DOK4 has been shown to be required for axon

myelination and the regulation of GDNF-dependent neurite outgrowth via the activation

of the Rap1-ERK1/2 pathway (Uchida et al., 2006). DOK5 was demonstrated to interact

with TrkB and TrkC neurotrophin receptors and is involved in the activation of the

MAPK pathway induced by neurotrophin stimulation (Shi et al., 2006). Like opioid

receptors, neurotrophins have a strong link to pain perception, reward, and synaptic

plasticity (Pardon, 2010; Pezet and McMahon, 2006). For example, BDNF (ligand of

63

TrkB) has been implicated in substance dependence as well as antinociception (Ghitza et

al., 2010; Marcol et al., 2007; Obata and Noguchi, 2006; Ren and Dubner, 2007). It will

certainly be of interest to determine whether there is functional interplay between the

MOR and neurotrophic factors in MAPK-mediated signaling, pain processing, reward,

and memory formation.

The results presented in this chapter highlight the use of multiple screening methods to

expand our knowledge of proteins that interact with, and may contribute to the regulation of

opioid receptor-mediated signaling in brain. Identification of the full panorama of MORIPs

represents a critical step in understanding the normal regulation of the receptor life-cycle, as well

as how receptor signaling or trafficking may be hijacked in response to drugs of abuse. The

MORIPs identified in this and other studies may also represent key players involved in other

opioid-mediated processes such as neural development, nociception, aversion, and synaptic

plasticity. As the list of MORIPs grows, and their contributions to receptor function become

better understood, it is possible that some of these proteins will also become targets for new drug

development to prevent and/or treat opioid addiction.

64

Chapter 3

Functional Analysis of the MOR/SIAH Interaction

3.1 Introduction

In Chapter 2, two E3 ligases, SIAH1 and SIAH2, were identified as novel

MORIPs. Since the identity of ubiquitin ligases that regulate the MOR are unknown,

these proteins were chosen for further study.

E3 ligases are the last enzymes utilized by the cell to modify proteins with

ubiquitin. Ubiquitin is attached to lysine residues of substrate proteins covalently and

requires the action of a cascade of enzymes. First, an ubiquitin-activating enzyme (E1)

forms an ATP-dependent high energy thiol ester bond with ubiquitin. Then the activated

ubiquitin is transferred to an ubiquitin-conjugating enzyme (E2). Some E2s are capable

of ubiquitinating the substrate directly; however, this is usually done by an ubiquitin

ligase (E3) and often confers substrate specificity (Myung et al., 2001).

Seven in absentia was first described as a gene required for the specification of R7

cell fate in the Drosophila melanogaster eye (Carthew and Rubin, 1990). Three years

later, the murine homologues were discovered and designated Siah (seven in absentia

homolog) and were found to exist as three genes, Siah-1a, Siah-1b, and Siah-2 (Della et

al., 1993). The first report and cloning of a human Siah gene suggested a role for Siah in

apoptosis and tumor suppression (Nemani et al., 1996). The human SIAH1 is

homologous to the mouse Siah1a, whereas the human SIAH2 is homologous to the mouse

Siah2. Humans do not have a homologus Siah1b gene (Hu et al., 1997a). The first report

65

of the involvement of Siah proteins in the ubiquitin-proteasome pathway was found when

the deleted in colorectal cancer (DCC) protein was shown to be regulated by Siah and

that Siah interacted with ubiquitin-conjugating enzymes (Hu et al., 1997b). Since these

initial studies, SIAH1 and SIAH2 have been shown to be RING (Really Interesting New

Gene)-finger E3 ligases capable of interacting with a variety of proteins and targeting

them for degradation via the ubiquitin-proteasome pathway.

Knockout mice have been generated for each of the Siah proteins. Knock out of

Siah1a (analogous to knock out of human SIAH1) causes postnatal growth retardation

and premature death, with few surviving beyond 3 months. Surviving males are infertile

and females are subfertile (Dickins et al., 2002). In contrast, Siah2 knockout mice do not

exhibit growth retardation or early mortality and both males and females are fertile, but

have abnormal bone marrow. Knock out of both Siah1 and Siah2 in combination results

in pups dying within hours of birth (Frew et al., 2003).

Both SIAH1 and SIAH2 have a protein-protein interacting domain in the C-

terminal region (amino acids 90-282 for SIAH1; amino acids 116-325 for SIAH2) and

the RING-finger (catalytic domain) in the N-terminal region (amino acids 1-89 for

SIAH1; amino acids 1-115 for SIAH2) (House et al., 2003; Polekhina et al., 2002). It has

been shown that these two domains can be separated, where mutations in the RING-

finger do not abrogate the binding of the proteins to their binding partners. The Siah

proteins have been shown to have auto-ubiquitinating activity and can form homo- and

heterodimers with each other and other E3 ligases (Hu and Fearon, 1999). They mediate a

variety of different pathways by regulating proteins via the ubiquitin-proteasome

pathway. Some of the reported targets of Siah-mediated degradation include

66

adenomatous polyposis coli protein (APC) (Liu et al., 2001), kinesin like DNA binding

protein (Kid) (Germani et al., 2000), synaptophysin (Wheeler et al., 2002), group 1

metabotropic glutamate receptor (Ishikawa et al., 1999), Pard3A (Famulski et al., 2010),

and beta-catenin (Dimitrova et al., 2010). SIAH2 has been reported to interact with Vav,

a GDP-exchange factor for Rac/Rho, but does not cause its degradation. SIAH2 was

shown to negatively regulate Vav-induced JNK activation (Germani et al., 1999). SIAH1

interacts with BAG1, an Hsp70/Hsc70-binding protein that modulates cell proliferation

and death, but does not cause its degradation. In fact, BAG1 inhibits the activity of

SIAH1 in p53-mediated cell cycle arrest (Matsuzawa et al., 1998). Therefore, not all

Siah-binding proteins are targets for Siah-mediated degradation.

In addition to the roles the Siah proteins play in protein-protein interactions and in

Siah-mediated degradation, these proteins have been implicated as players in

neurological disease and in addiction. Specifically, SIAH2 has been demonstrated to be

elevated in the blood of patients with psychosis and this elevation corresponded to greater

symptom severity (Bousman et al., 2010). SIAH1 has been shown to exhibit decreased

expression in the brains of patients with a history of alcohol abuse and/or dependence

although the functional consequence of this change was not pursued (Sokolov et al.,

2003).

The role of ubiquitin has largely been characterized to function as a sorting signal

for lysosomal degradation of GPCRs. Ubiquitin is best known to target agonist-activated

GPCRs to lysosomes for degradation via the endosomal-sorting complex required for

transport (ESCRT) pathway (Marchese et al., 2008). This process is important for

disposing of irreversibly activated receptors and for ridding cells of receptors after

67

chronic agonist stimulation (Marchese et al., 2008; Trejo et al., 1998). Interestingly,

delta opioid receptors are ubiquitinated following agonist stimulation, but ubiquitin is not

required for lysosomal sorting, indicating an additional undiscovered role for ubiquitin in

GPCR regulation (Hislop et al., 2009). More recent studies have demonstrated a role for

ubiquitination in the sorting of MORs from the endosome limiting membrane to the

lumenal compartment, but not in determining the recycling or degradation of the

receptors by the lysosome or proteasome (Hislop et al., 2011).

The identity of the ubiquitin ligases that mediate these responses is largely

unknown for the opioid receptors. However, an E3 ligase, atrophin-interacting protein 4

(AIP4), has been reported for the DOR. AIP4 was shown to control the down-regulation,

measured as loss of ligand binding, of DORs without affecting the delivery of the

receptor to lysosomes. This effect required the direct ubiquitination of receptors (Hislop

et al., 2009). The identity of E3 ligases that regulate either the KOR or the MOR have not

yet been identified.

Ubiquitination can serve as a signal for many cellular processes, including

degradation, trafficking, signal transduction, and endocytosis (Hicke and Dunn, 2003;

Marchese et al., 2008; Myung et al., 2001). Many of these same cellular processes

regulate the MOR and have been shown to play a role in the development of addiction

(Tuoc and Stoykova, 2010). E3 ligases are believed to confer substrate specificity for

ubiquitination; therefore, I investigated the functional role of the interaction between the

MOR and SIAH1. SIAH1 was chosen for further study over SIAH2, due to its reported

role in the ubiquitination and degradation of the metabotrophic glutamate receptor 1

(mGluR1), a GPCR (Moriyoshi et al., 2004). SIAH2 has no reported role in GPCR

68

regulation. Using overexpression and siRNA knockdown experiments, I show that

SIAH1 appears to form an interaction with SIAH2, and that cooperation between the Siah

proteins affects MOR ubiquitination and MOR protein levels.

3.2 Experimental Procedures

3.2.1 Mapping of binding sites

SIAH1 truncation (TR) deletion mutants (SIAH1-TR, residues 91-282; SIAH1-

TR2; residues 91-157, and SIAH1-TR3; residues 151-217) were ligated into pGEX-4T-1

to create GST fusion proteins. The PCR primers used in the cloning of the mutants can be

found in Table 3.1. The MORIL2-S-tag fusion protein was constructed as described in

Chapter 2. Fusion proteins were induced in Escherichia coli strain BL21 (DE3) using the

ZYP-5052 autoinduction media as described previously (De Cotiis et al., 2008; Studier,

2005). SIAH1 deletion mutant GST fusion proteins were used to pull down S-tagged

MORIL2 using glutathione sepharose beads (GE Healthcare) as previously described

(Lin et al., 2001). Eluted proteins were separated by SDS-PAGE and transferred to

polyvinylidene fluoride (PVDF) filters for western blot analysis. The filter was probed

with a horseradish peroxidase (HRP)-conjugated anti-S-tag antibody (1:5000 dilution,

Novagen, Madison, WI), and immunoreactivity was detected by enhanced

chemiluminescence with an ECL Plus kit (GE Healthcare).

69

Table 3.1: PCR primers used for cloning of SIAH1 deletion mutants

This table lists the PCR primers that were used to clone SIAH1 mutants into pGEX-4T-1

to produce GST-fusion proteins for use in GST pull down experiments.

Construct Direction Sequence

SIAH1-TR-GST

Amino Acids 91-282

Forward GGA TCC AAT TCA GTA CTT TTC CCC TGT

AAA

Reverse CTC GAG TCA ACA CAT GGC AAT AGT TAC

ATT GAT GCC

SIAH1-TR2-GST

Amino Acids 91-157

Forward GAA TTC AAT TCA GTA CTT TTC CCC TGT

AAA TAT

Reverse CTC GAG TAG GGT TGT AAT GGA CTT ATG

SIAH1-TR3-GST Forward GAA TTC AAG TCC ATT ACA ACC CTA CAG

Amino Acids 151-217 Reverse CTC GAG AAA ATT TTC AGC TTG CTT

3.2.2 Cell culture and transfections

Human embryonic kidney (HEK) 293 cells stably transfected with either FLAG-

tagged mu-opioid receptor (HEK-MOR), delta-opioid receptor (HEK-DOR), or kappa-

opioid receptor (HEK-KOR) were maintained in Dulbecco's Modified Eagle's Medium

(DMEM) supplemented with 10% fetal bovine serum (FBS) and 400 μg/mL G418. Full-

length SIAH1 and SIAH1 deletion mutant (SIAH1-TR, residues 91-282) were cut from

pGEX-4T-1 with EcoRI and XhoI restriction endonucleases and ligated into pCMV-

tag3B to create myc-tagged constructs. These constructs were transfected into HEK-

MOR cells using Effectene per manufacturer instructions (Qiagen, Valencia, CA).

70

3.2.3 Treatment of cells with inhibitors of the proteasome and lysosome

Human embryonic kidney (HEK) 293 cells stably transfected with FLAG-tagged

mu-opioid receptor (HEK-MOR) were treated for 30 minutes, 1 hour, 2 hours, and 4

hours with 50 μM cyclohexamide (protein synthesis inhibitor; Sigma) or 30 μM MG132

(proteasome inhibitor; Sigma). HEK-MOR cells were also treated for 30 minutes, 1 hour,

2 hours, 4 hours, and 6 hours with 50 μM of chloroquine (lysosome inhibitor; Sigma).

After the specified time point cells were collected and lysed in lysis buffer (20 mM

Na2HPO4, 20 mM NaH2PO4, 2 mM EDTA, 50mM sodium fluoride, 5 mM tetrasodium

pyrophosphate, 10mM β-glycerophosphate, 1% NP-40, 1 mM Pefabloc, 1 mM DTT, 1.2

mg/ml protease inhibitors). Crude cell lysates were run on SDS-PAGE and transferred to

PVDF membrane. To visualize the MOR blots were incubated with rabbit anti-FLAG

antibodies (1:500 dilution; Sigma) and goat anti-rabbit HRP-conjugated antibodies

(1:20,000 dilution; Jackson ImmunoResearch).

3.2.4 Detection of ubiquitinated MOR using TUBEs

HEK-MOR cells were transfected with SIAH1-myc or SIAH1-TR-myc as

described above. After 24 hours cells were lysed in lysis buffer containing 200 μg/mL of

tandem ubiquitin binding entities fused to GST (TUBEs; Life Sensors, Malvern, PA).

TUBEs contain multiple ubiquitin binding domains, which recognize and bind to

ubiquitin and protect the bound proteins from degradation via the proteasome. Cell lysate

was allowed to nutate for 30 minutes before the addition of glutathione sepharose beads

(Invitrogen, Grand Island, NY). Eluted proteins were resolved on SDS-PAGE and

71

transferred to PVDF membrane for western blotting. Blots were probed with rabbit anti-

MOR antibodies (1:5000 dilution AB5511; Millipore, Billerica, MA) and goat anti-rabbit

HRP conjugated antibodies (1:20,000 dilution; Jackson ImmunoResearch).

3.2.5 siRNA knockdown of SIAH proteins

HEK-MOR, HEK-DOR, or HEK-KOR cells were cultured in 100 cm2 plates to

50% confluency and then transfected with 120 pmol of Stealth siRNA for either SIAH1

(5’-GAUGCAUCAGCAUAAGUCCAUUACA-3’) or SIAH2 (5’-

ACUGGGUGAUGAUGCAGUCAUGUUU-3’) (Invitrogen) using RNAiMAX per

manufacturer instructions (Invitrogen). Cells were incubated for 72 hours and then

harvested in lysis buffer (20 mM Na2HPO4, 20 mM NaH2PO4, 2 mM EDTA, 50mM

sodium fluoride, 5 mM tetrasodium pyrophosphate, 10mM β-glycerophosphate, 1% NP-

40, 1 mM Pefabloc, 1 mM DTT, 1.2 mg/mL protease inhibitors) with 50μg/mL of

MG132 (Sigma, St. Louis, MO). MG132 was added to prevent protein degradation via

the proteasome during lysis. Western blotting was performed to confirm knockdown of

proteins.

3.2.6 Co-immunoprecipitation and western blot analysis

Crude cell lysate was prepared as described in siRNA knockdown of SIAH

proteins. Protein G magnetic agarose beads (GE Healthcare) incubated with polyclonal

rabbit anti-FLAG antibodies (Sigma) were used to immunoprecipitate receptors from

72

HEK-MOR, HEK-DOR, and HEK-KOR cell lysate. Immunoprecipitations (IPs) using

rabbit immunoglobulin g were performed as negative controls. IPs were resolved by

SDS-PAGE and transferred to PVDF membranes. For the detection of ubiquitin, blots

were first treated with 0.5% gluteraldehyde (as recommended by the antibody

manufacturer) (Sigma), then immunoblotted using mouse anti-ubiquitin antibody (1:5000

dilution of VU-1, LifeSensors) and rabbit anti-mouse HRP conjugated secondary

antibody (1:20,000 dilution, Jackson ImmunoResearch). The amount of endogenous

SIAH1 in crude lysate was determined by resolving lysates by SDS-PAGE and

immunoblotting using goat anti-SIAH1 (1:5000 dilution, Abnova, Taipei City, Taiwan)

antibody and bovine anti-goat HRP conjugated secondary antibody (1:20,000 dilution,

Jackson ImmunoResearch). The amount of SIAH2 in crude lysate was determined by

resolving lysates by SDS-PAGE , treating blots with Pierce Western Blot Enhancer Kit

per manufacturer instructions (Pierce), and immunoblotting using goat anti-SIAH2

antibody (1:500 dilution, N-14, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and

bovine anti-goat secondary antibody (1:20,000 dilution, Jackson ImmunoResearch). The

amount of opioid receptors in crude lysate was determined by immunoblotting with rabbit

anti-FLAG antibody (1:5000 dilution, Sigma) and goat anti-rabbit HRP conjugated

antibody (1:20,000 dilution, Jackson ImmunoResearch). To control for loading, blots

were also probed for GAPDH (1:5000 dilution, Sigma) using chicken anti-GAPDH

antibodies and donkey anti-chicken antibody (1:20,000 dilution, Jackson

ImmunoResearch) or tubulin (1:10,000 dilution, Novus) using mouse anti-tubulin

antibodies and goat anti-mouse HRP conjugated antibody (1:20,000 dilution, Jackson

ImmunoResearch).

73

3.2.7 Quantitation of Western blots

All blots were run in duplicate or triplicate. Blots were scanned using a back-lit

scanner and quantitation was performed using ImageJ software (Abramoff et al., 2004).

Expression was normalized to total protein for siRNA experiments (as measured by

GAPDH), averaged between replicates, and subjected to a two-tailed Student’s t-test.

3.3 Results

3.3.1 Mapping of the MOR binding site on SIAH1

In Chapter 2, the binding site for SIAH1 on the MOR was found to be the second

intracellular loop. However, the location where MORIL2 binds to SIAH1 has not been

determined. The SIAH1 protein can be divided into an N-terminal region (residues 1-

89), which includes the RING domain (residues 40-75), and the C-terminal region (amino

acids 90-282), which is involved in substrate binding (Hu and Fearon, 1999). The RING

domain is the catalytic domain of the protein and is required for ubiquitination of target

proteins (Hu and Fearon, 1999). I therefore hypothesized that the C-terminal region of

SIAH1 would bind to the second intracellular loop of the MOR. To determine a more

specific binding region, SIAH1 deletion mutants were made in the C-terminal region of

the protein (Figure 3.1B). As expected, the C-terminal region but not the N-terminal

region of SIAH1 was required for SIAH1 to bind to the MOR (Figure 3.1A). Upon the

truncation of the C-terminal region, amino acids 91-157 (SIAH1-TR2-GST) were able to

74

pull down MORIL2, while amino acids 151-282 (SIAH1-TR3-GST) were no longer able

to pull down MORIL2 (Figure 3.1A). This indicates that the region of the C-terminus

that includes amino acids 91-157 is required for the interaction between SIAH1 and

MORIL2.

Figure 3.1: Mapping of SIAH1 binding site

(A) Deletion mutants of SIAH1 were used in GST pull down experiments to localize the

MOR binding site on SIAH1. SIAH1-TR-GST (a. a 91-282), SIAH1-TR2-GST (a. a 91-

157), and SIAH1-TR3-GST (a. a 151-217) were used to pull down S-tagged MORIL2.

Pull-down products were purified on glutathione beads, separated by SDS-PAGE, and

probed on western blot using HRP-conjugated anti-S-tag antibodies. S-tagged MORIL

produced in bacteria are shown in the lysate lane (LY), while uncoated glutathione

sepharose beads (BEADS) or GST-coated beads (GST) incubated with S-tagged

MORIL2 served as controls. (B) Map of constructs. The green represents the N-terminal

region, which includes the RING-finger domain in gold (RF). The pink represents the C-

terminal region, which includes the substrate binding domain in pink (SBD).

75

3.3.2 SIAH1 regulates the amount of MOR modified with ubiquitin

As an E3 ligase, SIAH1 has the ability to conjugate ubiquitin to its binding

partners. I wanted to determine if SIAH1 changes the ubiquitination of the MOR. To

test this, HEK-MOR cells were transfected with SIAH1-myc or SIAH1-TR-myc.

SIAH1-TR-myc has the RING domain (catalytic domain) removed and should not be

able to participate in ubiquitination, but retains the ability to bind to the MOR. Tandem

ubiquitin binding entities (TUBEs) fused to GST were used to pull down the total

ubiquitinated protein from cell lysates. HEK-DOR cells were mock transfected and used

as a negative control to demonstrate the specificity of the MOR antibody (Figure 3.2 A).

MOR protein modified with ubiquitin often resolves as a broad high molecular weight

band or smear of 75 -250 kDa on western blot (Groer et al., 2011). Due to this broad

band, quantitation cannot be performed for this kind of analysis and is not performed by

other researchers in the field. In HEK-MOR cells transfected with empty vector, the pull

down lane shows ubiquitinated MOR as an immunoreactive band between 100 and 200

kDa (Figure 3.2, top panel, lane 2). Unexpectedly, when SIAH1 was overexpressed, the

intensity of the immunoreactive band between 100 and 200 kDa decreased slightly

(Figure 3.2 A, lane 4). This indicates that overexpression of SIAH1 may cause a

reduction in the amount of MOR that is ubiquitinated. In contrast, when the RING

mutant was overexpressed, the intensity of this band increased (Figure 3.2 A, lane 6).

To confirm the results seen with the TUBEs, I next immunoprecipitated the MOR

and probed the eluted material for ubiquitin. If SIAH1 is an E3 ligase for the MOR, the

overexpression of SIAH1 would be expected to cause the amount of ubiquitinated MOR

76

to increase. Although not as robust as the TUBEs experiment, the amount of MOR that is

ubiquitinated is slightly decreased when SIAH1 is overexpressed as compared to mock

transfected cells (Figure 3.2B, top panel, lanes 3 and 5). Conversely, when the RING

mutant SIAH1 is overexpressed, there is a slight increase in the amount of ubiquitinated

MOR (Figure 3.2B, top panel, lane 7). Without the ability to quantitate these results, it is

difficult to determine the significance of the effect of SIAH1 on ubiquitination of the

MOR. However, SIAH1-TR overexpression did cause a noticeable increase in the

ubiquitination of the MOR in the TUBEs experiment, which indicates that the RING-

finger is not responsible for the increase in the observed ubiquitination. Therefore,

SIAH1 is likely not an E3 ligase for MOR.

77

Figure 3.2: SIAH1 decreases the ubiquitination of the MOR

(A) HEK-MOR cells were transfected with empty vector (pCMV-tag3B), SIAH1 with a

myc tag (SIAH1-myc), SIAH1 RING mutant with a myc tag (SIAH1-TR-myc). The

amount of MOR eluted from the beads was determined by immunoblotting with anti-

MOR antibodies and is shown in the IP lanes. HEK-DOR cells were used as a control to

show that the TUBEs do not cause non-specific banding patterns. (B) HEK-MOR cells

were transfected as in (A) and cell lysate was subjected to immunoprecipitation with anti-

FLAG antibodies to IP the MOR and then immunoblotted with mouse anti-ubiquitin

antibodies. IPs using rabbit IgG was used as a negative control (M). Crude cell lysates

were immunoblotted to show that the constructs were expressed in the cells used in pull

down or immunoprecipitation experiments (Bottom panels A and B). Blots are

representative of three separate experiments.

78

3.3.3 siRNA knockdown of SIAH1 increases the ubiquitination of the opioid receptors

Since the overexpression of SIAH1 caused a decrease in the amount of detectable

ubiquitinated MOR, I expected that knocking down endogenous SIAH1 would result in

increased ubiquitination of MOR. HEK-MOR cells were transfected with siRNA against

SIAH1. Crude cell lysate was immunoblotted to detect the amount of SIAH1 and

GAPDH (Figure 3.3B). Knockdown of SIAH1 resulted in an 80% reduction in the

amount of endogenous Siah1 protein (Figure 3.3C). To determine the ubiquitination

level of the MOR, MOR was immunoprecipitated from lysates of cells that were

transfected with scrambled or SIAH1 siRNA, subjected to SDS-PAGE, and

immunoblotted with anti-ubiquitin antibodies. As expected, Siah1 knockdown resulted in

an increase in the amount of detectable ubiquitinated MOR (Figure 3.3A). These results

are consistent with observations made by overexpression of SIAH1-TR-myc, which

suggests that SIAH1-TR-myc acts as a dominant negative mutant. It is possible that this

assay is not measuring MOR ubiquitination, but the ubiquitination of another MORIP.

This assay is indirect as any protein that immunoprecipitates with the MOR and is also

ubiquitinated may be observed on the blot. However, this assay can be used to determine

whether or a particular protein is a putative E3 ligase or not. If SIAH1 is an E3 ligase for

the MOR, its knockdown should cause the ubiquitination of the MOR to decrease, not

increase. Therefore, SIAH1 most likely is not an E3 ligase for the MOR.

The opioid receptors show considerable similarity in the second intracellular loop

(Chapter 2, Figure 2.3A) and SIAH1 has been shown to co-immunoprecipitate with all

three receptors (Chapter 2, Figure 2.3B). Therefore, it seems plausible that SIAH1 might

79

affect the ubiquitination of DOR and/or KOR. To investigate this possibility, siRNA

against SIAH1 was transfected into HEK-DOR cells and HEK-KOR cells. Siah1 protein

was knocked down by 94% in HEK-DOR cells and by 82% in HEK-KOR cells (Figures

3.3B and 3.3C). As with the MOR, knockdown of SIAH1 caused an increase in the

detectable ubiquitination of DOR and KOR (Figure 3.3A). These results indicate that

SIAH1 is likely not an E3 ligase for either DOR or KOR.

80

Figure 3.3: Knockdown of SIAH1 increases ubiquitination of MOR, DOR, and

KOR

HEK-MOR, HEK-DOR, and HEK-KOR cells were transfected with siRNA against

SIAH1 (KD) or scrambled siRNA (SC). (A) Cells were lysed and the opioid receptors

were immunoprecipitated using rabbit anti-FLAG antibodies. Blots were probed with

mouse anti-ubiquitin. The ubiquitinated opioid receptors yield immunoreactive smears

between 75-200 kDa. IPs using normal rabbit IgG were used as a negative control

(Mock). (B) Western blots showing the amount of SIAH1 in lysate was used to confirm

the knockdown, while GAPDH was used as a loading control. (C) Western blot bands

were quantitated using ImageJ and normalized to GAPDH. Data was analyzed using a

paired Student’s t-test expressed as mean ± SEM; n=3, *p < 0.05).

81

3.3.4 Knockdown of SIAH2 results in decreased ubiquitination of MOR, but increased

ubiquitination of DOR and KOR.

Another E3 ligase, SIAH2, was found to interact with all three opioid receptors

(Chapter 2). Since SIAH1 did not seem to be a candidate E3 ligase for any of the opioid

receptors, I wanted to determine if SIAH2 had any effect on the ubiquitination of the

opioid receptors. To test this HEK-MOR, HEK-DOR, and HEK-KOR cells were

transfected with siRNA targeting SIAH2.

Crude cell lysate was immunoblotted to detect the amount of SIAH2, and

GAPDH (Figure 3.4B). Knockdown of SIAH2 resulted in a 98% reduction in the amount

of endogenous Siah2 protein (Figure 3.4C). To determine the ubiquitination level of the

MOR, MOR was immunoprecipitated from lysates of cells that were transfected with

scrambled or SIAH2 siRNA, subjected to SDS-PAGE, and immunoblotted for ubiquitin.

SIAH2 knockdown resulted in a slight decrease in the amount of detectable ubiquitinated

MOR (Figure 3.4A). These results are more consistent with SIAH2 being an E3 ligase

involved in MOR ubiquitination because the ubiquitination of MOR would be expected

to decrease. Therefore, SIAH2 may be a putative E3 ligase of the MOR.

Siah2 protein was knocked down by 96% in HEK-DOR cells and 97% in HEK-

KOR cells (Figuress 3.4B and 3.4C). In contrast to the MOR, knockdown of SIAH2

caused an increase in the detectable ubiquitination of DOR and KOR (Figure 3.4A). If

SIAH2 is an E3 ligase for the DOR and/or KOR, the knockdown would be expected to

result in a decrease in the ubiquitination of DOR. Therefore, these results indicate that

SIAH2 is most likely not an E3 ligase for this receptor. However, if the results of SIAH1

and SIAH2 knockdown in HEK-DOR and HEK-KOR cells are considered together, they

82

suggest that the Siah proteins may compensate for each other and therefore, cannot be

ruled out as putative E3 ligases for these receptors.

Figure 3.4: Knockdown of SIAH2 decreases ubiquitination of MOR, but increases

ubiquitination of DOR and KOR

HEK-MOR, HEK-DOR, and HEK-KOR cells were transfected with siRNA against

SIAH2 (KD) or scrambled siRNA (SC). (A) Cells were lysed and the opioid receptors

were immunoprecipitated using rabbit anti-FLAG antibodies. Blots were probed with

mouse anti-ubiquitin. IPs using rabbit IgG were used as negative controls (Mock). The

ubiquitinated opioid receptors yield immunoreactive smears between 75-200 kDa. (B)

Western blots showing the amount of SIAH2 in cell lysate was used to confirm the

knockdown, while GAPDH was used as a loading control. (C) Western blot bands were

quantitated using ImageJ and normalized to GAPDH. Data was analyzed using a paired

Student’s t-test expressed as mean ± SEM; n=3, *p < 0.05).

83

3.3.5 The MOR undergoes degradation via the proteasome and lysosome

The degradation of the MOR has been demonstrated to occur primarily through

the lysosomal pathway, but has also been demonstrated to occur via the proteasome

pathway in overexpression cell culture systems. However, both pathways have been

observed in to occur in overexpression cell culture systems (Chaturvedi et al., 2001;

Hislop and von Zastrow, 2011). Since E3 ligases can target proteins to either pathway in

the HEK-MOR cells, I wanted to determine if the MOR was capable of undergoing

degradation via the proteasome and/or the lysosome. This was tested by treating HEK-

MOR cells with cyclohexamine, MG132, or chloroquine for varying amounts of time. A

protein that undergoes proteasomal degradation should accumulate in the cell as the

length of proteasome inhibitor, MG132, exposure increases. Likewise, a protein that

undergoes lysosomal degradation should accumulate in the cell as the length of lysosomal

inhibitor, chloroquine, exposure increases. Cyclohexamide, a protein synthesis inhibitor,

was used to determine the half-life of the MOR. The levels of a protein that are

degraded (lysosomal or proteasomal) will decrease in the cell overtime if protein

synthesis is inhibited. The MOR has previously been shown to resolve on Western blot

as two major bands, one at 50 kDa and one at 75 kDa due to varying amounts of

glycosylation (Huang and Liu-Chen, 2009). Interestingly, only the low molecular weight

(50 kDa band) form of the MOR was observed to change with either cyclohexamide

(Figure 3.5A) or MG132 (Figure 3.5B) treatment. The half-life of the low molecular

MOR band is short, as this band is no longer detectable after 1 hour of cyclohexamide

treatment (Figure 3.5A; 50 kDa band). This result suggests that the low molecular weight

84

form of MOR is either subject to degradation or further post-translational modification

(perhaps maturing into high molecular weight form of MOR). It is likely that a portion

of low molecular weight MOR is subject to proteasomal degradation because the levels

of this protein species increased with lengthening MG132 exposure (Figure 3.5B; 50 kDa

band). The high molecular weight form of the MOR (75 kDa band) did not change with

either cyclohexamide or MG132 treatment (Figures 3.5A and B) indicating that this

protein species is stable and not degraded within the time points measured. Therefore, no

conclusions on the involvement of the proteasome in degradation of the high molecular

weight form of MOR can be made from this experiment.

Contrary to the results seen with the proteasome block, both the low molecular

weight band (50 kDa band) and the high molecular weight band (75 kDa band) of MOR

increased over time with chloroquine treatment (Figure 3.5C). Thus, it appears that

lysosomal degradation has a more prominent role than proteasomal degradation for the

MOR.

85

Figure 3.5: Inhibitor treatment of HEK-MOR cells

HEK-MOR cells were treated with (A) 50 μM cyclohexamide, a protein synthesis

inhibitor, (B) 30 μM MG132, a proteasome inhibitor, or (C) 50 μM of chloroquine, a

lysosomal inhibitor for 30 minutes, 1 hour, 2 hours, and 4 hours. Cells treated with

chloroquine were also treated for 6 hours. Cells were treated with vehicle (0) as a

control. After treatment cells were lysed and crude cell lysate was resolved. Blots are

representative of three individual experiments.

3.3.6 Overexpression of SIAH1 causes a decrease in MOR protein levels

Since both the proteasomal and lysosomal degradation pathways can be mediated

by ubiquitination, I wanted to determine the effects of overexpressing SIAH1 on MOR

protein levels in HEK-MOR cells. Although it appears that SIAH1 does not ubiquitinate

the MOR, it may still play a role in its degradation. If SIAH1 is involved in MOR

degradation by either pathway, overexpression of the protein would be expected to cause

the amounts of detectable MOR to decrease. HEK-MOR cells were transfected with full-

length SIAH1 fused to myc using increasing microgram concentrations of the SIAH1-

myc construct or with vector as a negative control. Transfecting increasing amounts of

SIAH1-myc resulted in decreasing amounts of both the low molecular weight (50 kDa

86

band) and the high molecular weight (75 kDa band) species of MOR protein with

increasing concentrations of SIAH1-myc (Figure 3.6). These results indicate that SIAH1

may regulate MOR protein levels in mammalian cells.

Figure 3.6: SIAH1 induces MOR degradation

HEK-MOR cells were transfected with increasing amounts of SIAH1-myc DNA. Vector

DNA (V) was added to make the total amounts of DNA transfected equal. 2.0 μg of

vector DNA was transfected as a negative control. The amount of MOR-FLAG, SIAH1-

myc, and GAPDH in cell lysates was determined by immunoblotting with anti-FLAG,

anti-myc, and anti-GAPDH antibodies, respectively. GAPDH was used as a loading

control. Blot images are representative of three experiments.

3.3.7 siRNA knockdown of SIAH1 increases MOR protein levels, but decreases DOR and

KOR protein

Since the overexpression of SIAH1 caused a decrease in MOR protein levels, I

expected that knocking down endogenous SIAH1 would result in increased MOR protein

levels. To test this, siRNA against SIAH1 was transfected into HEK-MOR cells. The

reduction in Siah1 protein levels caused a 16-fold increase in the amount of low

87

molecular weight MOR (50 kDa band) (Figures 3.7A and D), while no significant change

was detected for the high molecular weight version of MOR (75 kDa band) (Figures 3.7A

and 3.7C). This suggests that SIAH1 may play a role in the regulation of MOR protein

levels.

Since SIAH1 had been shown to co-immunoprecipitate with all three receptors,

and knockdown of SIAH1 caused an increase in ubiquitination of the DOR and KOR, it

seems possible that SIAH1 might affect DOR and/or KOR protein levels. To investigate

this possibility, siRNA against SIAH1 was transfected into HEK-DOR cells and HEK-

KOR cells. In contrast to the results for MOR, the levels of both the DOR low molecular

weight protein (45 kDa band) (Figures 3.7A and D) and the high molecular weight DOR

(55 kDa band) decreased with knockdown of SIAH1 (Figuress 3.7A and 3.7C). The

DOR is also differentially glycosylated and the observation of multiple bands is due to

this modification (Petaja-Repo et al., 2000). The levels of KOR low molecular weight

protein (45 kDa) (Figuress 3.7A and 3.7D) and high molecular weight protein (55 kDa)

(Figures 3.7A and 3.7C) also decreased, as was the case with DOR. Like the MOR and

the DOR these differences in molecular weight are due to glycosylation (Chen et al.,

2006). Therefore, these results indicate that SIAH1 may play a role in the regulation of

DOR and KOR protein levels.

88

Figure 3.7: Knockdown of SIAH1 changes MOR, DOR, and KOR protein levels

HEK-MOR, HEK-DOR, and HEK-KOR cells were transfected with siRNA against

SIAH1 (KD) or scrambled siRNA (SC). (A) Western blots showing the levels of

receptor in cell lysate. (B) Western blot showing the levels of SIAH1 protein to confirm

the knockdown. GAPDH was used as a loading control. (C) High molecular weight

bands on Western blots were quantitated using ImageJ and normalized to GAPDH. Data

was analyzed using a paired Student’s t-test expressed as mean ± SEM; n=3, *p < 0.05).

(D) Low molecular weight bands on Western blots were quantitated as in (C).

89

3.3.8 siRNA knockdown of SIAH2 decreases MOR protein levels, but increases DOR and

KOR protein

Since SIAH2 knockdown resulted in decreased ubiquitination of the MOR, I

wanted to determine if SIAH2 was involved in degradation of the receptor. To test this,

HEK-MOR cells were transfected with siRNA targeting SIAH2. The reduction in Siah2

protein levels caused a significant decrease in the amount of both the low molecular

weight MOR (band of 50 kDa) (Figures 3.8A and 3.8D) and the high molecular weight

MOR (band of 75 kDa) (Figures 3.8A and 3.8C). However, if SIAH2 is the E3 ligase

that targets the MOR for degradation, the knockdown of Siah2 protein should result in an

increase in MOR protein levels. Therefore, SIAH2 may be a putative E3 ligase of the

MOR, but its tagging of the MOR with ubiquitin may not result in degradation of the

MOR.

In contrast to the results for MOR, the ubiquitination of DOR and KOR increased

with SIAH2 knockdown. Therefore, I wanted to determine if this ubiquitination lead to

degradation of these receptors. Knockdown of Siah2 protein, caused the levels of the

DOR low molecular weight protein (45 kDa band) (Figures 3.8A and 3.8D) to increase

significantly, but did not significantly change the high molecular weight DOR (55 kDa

band) protein levels (Figures 3.8A and3.8C). Contrary to the results seen in HEK-DOR

and HEK-MOR cells, the levels of the KOR low molecular weight protein (45 kDa band)

(Figures 3.8A and 3.8D) and the high molecular weight KOR (55 kDa band) both

significantly increased with knockdown of SIAH2 (Figures 3.8A and 3.8C). Together

these results suggest that SIAH2 may play a role in the degradation of DOR and KOR.

90

Figure 3.8: Knockdown of SIAH2 changes MOR, DOR, and KOR protein levels

HEK-MOR, HEK-DOR, and HEK-KOR cells were transfected with siRNA against

SIAH1 (KD) or scrambled siRNA (SC). (A) Western blots showing the levels of

receptor in cell lysate. (B) Western blots showing the levels of SIAH1 protein to confirm

the knockdown. GAPDH was used as a loading control. (C) High molecular weight

bands on Western blots were quantitated using ImageJ and normalized to GAPDH. Data

was analyzed using a paired Student’s t-test expressed as mean ± SEM; n=3, *p < 0.05).

(D) Low molecular weight bands on Western blots were quantitated as in (C).

91

3.3.9 SIAH1 and SIAH2 interact in HEK-MOR cells

If SIAH1 is an E3 ligase for the MOR, its overexpression would be expected to

cause an increase in the amount of ubiquitinated receptor; however, this was not the case.

In Chapter 2, SIAH2 was also identified as an interactor with the MOR. The Siah1 and

Siah2 proteins share more than 85% amino acid identity in the C-terminal region (amino

acids 80-324) and 77% overall identity. Essentially, they only differ from one another in

the length and sequence of the extreme N-terminus, which includes a portion of the ring

domain (Hu et al., 1997b). Therefore, the antibodies used in this study were to epitopes

in the N-terminus of the proteins. There is evidence that SIAH1 and SIAH2 can form

heterodimers with each other and in some cases regulate each other’s function (Depaux et

al., 2006; Hu and Fearon, 1999). Additionally, the Siah proteins have been shown to

interact with some of the same proteins, such as ALL-1 fused gene on chromosome 4

(Af4) (Bursen et al., 2004; Oliver et al., 2004), phospholipase C eplison (Yun et al.,

2008) and alpha synuclein (Szargel et al., 2009). Since SIAH2 was also found to be a

MOR interactor, I wanted to determine if SIAH1 and SIAH2 interact in HEK-MOR cells

as this may affect ubiquitination and degradation studies of MOR. Due to the lack of

commercial antibodies that can immunoprecipitate either Siah protein, HEK-MOR cells

were transfected with SIAH2-myc. Anti-myc antibodies were used to immunoprecipitate

SIAH2-myc and anti-SIAH1 antibodies were used to detect SIAH1 in eluted proteins via

western blot. As shown in the IP lane of Figure 3.9, SIAH2-myc was able to co-

immunoprecipitate with SIAH1. These results indicate that SIAH1 and SIAH2 may

interact with each other in HEK-MOR cells.

92

Figure 3.9: Co-immunoprecipitation of SIAH1 with SIAH2

HEK-MOR cells were transfected with SIAH2-myc. Myc-tagged SIAH2 was

immunoprecipitated using mouse anti-myc antibodies. Mock immunoprecipitations were

performed with Protein-G beads coated with normal rabbit IgG. Blot was probed with

goat anti-SIAH1 antibodies. Lysate lane contains 5% of the total protein compared to the

mock (M) and immunoprecipitation lands (IP).

3.3.10 SIAH1 regulates the levels of SIAH2 in HEK cells

After determining that SIAH1 and SIAH2 can interact in HEK-MOR cells, I

wanted to determine if either Siah protein had an effect on the protein level of the other.

Knockdown of SIAH1 resulted in a two-fold increase in Siah2 protein in HEK-MOR,

HEK-DOR, and HEK-KOR cells (Figures 3.10A and 3.10B). This result was somewhat

unexpected, as SIAH2 has been shown to be capable of regulating SIAH1 levels, but the

opposite has not (Depaux et al., 2006). However, my results suggest that SIAH1

regulates SIAH2 levels.

93

Figure 3.10: SIAH1 knockdown causes an increase in SIAH2 protein levels

HEK-MOR, HEK-DOR, and HEK-KOR cells were transfected with siRNA against

SIAH1 (KD) or scrambled siRNA (SC). (A) Western blots showing levels of SIAH1 (to

confirm knockdown), SIAH2, and GAPDH (loading control). (B) Western blots were

quantitated using ImageJ and normalized to GAPDH. Data was analyzed using a paired

Student’s t-test expressed as mean ± SEM; n=3, *p < 0.05).

Although SIAH1 appears to regulate the levels of SIAH2 protein, the knockdown

of SIAH2 did not result in an increase in SIAH1 protein levels in any of the cell lines

tested (Figures 3.11A and 3.11B). Again this was unexpected as SIAH2 has been shown

to regulate SIAH1 levels (Depaux et al., 2006). However, it does not appear that SIAH2

regulates the protein level of SIAH1.

94

Figure 3.11: Knockdown of SIAH2 does not alter SIAH1 protein levels

HEK-MOR, HEK-DOR, and HEK-KOR cells were transfected with siRNA against

SIAH2 (KD) or scrambled siRNA (SC). (A) Western blots showing levels of SIAH2 (to

confirm knockdown), SIAH1, and GAPDH (loading control). (B) Western blots were

quantitated using ImageJ and normalized to GAPDH. Data was analyzed using a paired

Student’s t-test expressed as mean ± SEM; n=3, *p < 0.05).

3.3.11 Knockdown of both SIAH1 and SIAH2 results in a decrease in the ubiquitination of

the MOR and an increase in MOR protein levels

Knockdown of an E3 ligase involved in the ubiquitination and subsequent

degradation of the MOR would be expected to cause both a decrease in ubiquitination of

the MOR and an increase in MOR protein levels. Since neither SIAH1 nor SIAH2

knockdown alone had that effect on the MOR, I wanted to determine the effect of a

double knockdown on protein levels and ubiquitination. HEK-MOR cells were

95

transfected with siRNA targeting SIAH1 and SIAH2 in combination. There were

difficulties in knocking both proteins in combination; therefore the experiments were

performed in duplicate and no statistical analysis was performed. However, it is clear

that SIAH1 protein was knocked down, as was SIAH2 (Figure 3.12B). Due to the lack of

replicates, the effect of the double knockdown on MOR protein levels cannot be

determined. Interestingly, the detection of ubiquitinated MOR was abolished when both

SIAH1 and SIAH2 were knocked down in combination (Figure 3.12A). When these

results are considered with the results of the knockdown of the Siah proteins singly, they

indicate that the Siah proteins may cooperate to regulate the levels of MOR protein and

the ubiquitination the MOR.

96

Figure 3.12: Knockdown of both SIAH1 and SIAH2 decreases MOR ubiquitnation

HEK-MOR cells transfected with siRNA against SIAH1 and SIAH2 in combination

(DKD) or scrambled siRNA as a control (SC). (A) Crude cell lysate was

immunoprecipitated with rabbit anti-FLAG antibodies and probed with mouse anti-

ubiquitin antibodies to detect ubiquitinated MOR. (B) Cell lysate was immunoblotted

with rabbit anti-FLAG antibodies, goat anti-SIAH1 and goat anti-SIAH2 to detect MOR,

SIAH1, and SIAH2, respectively. GAPDH was used as a loading control.

3.4 Discussion

The functional relevance of the SIAH1/MOR interaction was investigated using a

series of experiments designed to test the hypothesis that SIAH1 is the E3 ligase that

mediates the ubiquitination and subsequent degradation of the MOR. If this hypothesis is

97

correct, overexpression of SIAH1 should result in a decrease in MOR protein levels and

MOR ubiquitination. In line with this hypothesis, when myc-tagged SIAH1 was

transfected in increasing concentrations, the amount of MOR decreased. However, this

observed decrease was found to be different for the two species of MOR detected on

Western blots. The high molecular weight species (band of 75 kDa) was not as sensitive

to the effect of SIAH1 overexpession as the low molecular weight species (band of 50

kDa). These differences in molecular weight have been reported to be attributable to the

amount of N-linked glycosylation present on the receptor as the predicted size of the

unmodified MOR protein is approximately 43 kDa. These two species of the receptor

correspond to highly glycosylated plasma membrane associated receptors (60-100 kDa)

and slightly glycosylated or unglycosylated intracellularly associated (40-60 kDa)

receptors (Huang and Liu-Chen, 2009). These results indicate that SIAH1 may be acting

on the immature form of the MOR, possibly at the endoplasmic reticulum or some other

intracellular location.

Treatment of HEK-MOR cells with the proteasome inhibitor, MG132, shows that

the intracellular MOR (low molecular weight, 50 kDa band) is sensitive to degradation

via the proteasome, but that the plasma membrane MOR is not (high molecular weight,

75 kDa band) within the time frame tested. These results are consistent with recent

evidence showing that the plasma membrane MOR is degraded via the lysosome and that

ubiquitination does not serve as a signal for degradation via the proteasome (Hislop et al.,

2011). When HEK-MOR cells were treated with the lysosomal inhibitor, chloroquine,

both species of the receptor increased over time. Taken together, these results suggest

98

that the immature, intracellular form of the MOR is sensitive to proteasomal degradation

and that this degradation may be mediated by SIAH1.

Contrary to the proposed hypothesis, overexpression of myc-tagged SIAH1

caused a decrease in the amount of ubiquitinated MOR in both the TUBEs experiments

and MOR immunoprecipitation experiments, while overexpression of a RING-deficient

mutant (SIAH1-TR-myc) caused an increase in detectable ubiquitinated MOR. Although

these results are contradictory to the hypothesis that SIAH1 is an E3 ligase for MOR,

they are confirmed by the SIAH1 siRNA knockdown experiments in HEK-MOR cells

and suggest that the RING domain is not required for this increase in ubiquitination of the

MOR. A summary of results from the overexpression and siRNA knockdown

experiments is shown in Table 3.2. The siRNA knockdown of SIAH1 and the

overexpression of myc-tagged RING-deficient SIAH1 show similar results which is

consistent with the previous observation that the RING domain mutant functions as

dominant negative (Hu and Fearon, 1999). Both the knockdown of SIAH1 and the

overexpression of SIAH1-TR-myc resulted in an increase in the amount of ubiquitinated

MOR, which is not in line with SIAH1 playing a role as an E3 ligase which conjugates

ubiquitin to the MOR.

99

Table 3.2: Summary of SIAH overexpression and knockdown experiments

For experiment column, upward arrows indicate overexpression, whereas downward

arrows indicate siRNA knockdown. For effect on Siah proteins, ubiquitination, and

protein levels, upward arrows indicate an increase and downward arrows indicate a

decrease. ND indicates that the experiment was not performed.

Cell Line Experiment Effect on Siah

proteins

Effect on

ubiquitination

Effect on

protein levels

HEK-MOR SIAH1 ↑ ND ↓ Both species ↓

HEK-MOR SIAH1-TR ↑ ND ↑ ND

HEK-MOR SIAH1↓ SIAH2 ↑ ↑ Low species ↑

HEK-MOR SIAH2 ↓ No change in

SIAH1

↓ Both species ↓

HEK-MOR SIAH1 ↓ and

SIAH2 ↓

Not applicable ↓ Low species ↑

HEK-DOR SIAH1 ↓ SIAH2 ↑ ↑ Both species ↓

HEK-DOR SIAH2 ↓ No change in

SIAH1

↑ Low species ↑

HEK-KOR SIAH1 ↓ SIAH2 ↑ ↑ Both species ↓

HEK-KOR SIAH2 ↓ No change in

SIAH1

↑ Both species ↑

The knockdown of SIAH1 caused a significant increase in the amount of another

MORIP, SIAH2, which is an E3 ligase that can interact with SIAH1 and MOR in these

cells. One could argue that the results obtained in the SIAH1 knockdowns could be due

to the upregulation of SIAH2. This is partially supported by the observation that MOR

ubiquitination and protein expression levels are reduced in SIAH2 knockdowns in HEK-

MOR cells, a result that is opposite from those obtained from SIAH1 knockdown.

However, when both SIAH1 and SIAH2 were knocked down in combination in HEK-

MOR cells, the amount of detectable ubiquitinated MOR was abolished.

In summary, the results obtained from the knockdown of either SIAH1 and

SIAH2 alone are not consistent with the hypothesis that one of these individually acts as

100

both an E3 ligase and an inducer of degradation for the MOR. On the contrary, a

combinatorial knockdown of SIAH1 and SIAH2 did provide results that would be

consistent with the hypothesis that these two proteins may work in concert to ubiquitinate

MOR. Unfortunately, no conclusions can be drawn about the effect of the double

knockdown on the levels of MOR protein as not enough replicates were able to be used

for statistical analysis.

The effect of Siah protein knockdown on the amount of low and high molecular

MOR species is unexpected. SIAH1 knockdown caused the amount of the low molecular

weight (50 kDa band), intracellular MOR to increase significantly but did not

significantly affect the high molecular weight (75 kDa band), plasma membrane-

associated form of MOR (Huang and Liu-Chen, 2009). This suggests that SIAH1 may

have a role in the regulation of intracellular MOR protein levels, but does not

significantly affect MOR at the plasma membrane. In contrast, SIAH2 knockdown

caused a decrease in both species. The knockdown of SIAH2 may allow the activity of

SIAH1 to increase, but this increased activity is not due to increases in Siah1 protein

levels, as the amount of Siah1 protein did not increase as shown via Western blot

analysis. Alternatively, SIAH2 may regulate another protein that is involved in

regulation of MOR, resulting in decreased levels of MOR protein. However, the results

of the double knock down experiments indicate that the Siah proteins are working

together, as there is a reduction in the ubiquitination of MOR. These results also indicate

that SIAH2 may participate in ubiquitination of the MOR, but that SIAH1 is required for

degradation of the receptor. Models for the role of the Siah proteins in the ubiquitination

and regulation of the MOR are discussed in more detail in Chapter 5.

101

Knockdown of either SIAH1 or SIAH2 in HEK-DOR or HEK-KOR cells resulted

in an increase in the ubiquitination of DOR or KOR, respectively. These results may

indicate that both Siah proteins play a role in the ubiquitination of DOR and KOR.

Additionally, SIAH1 knockdown caused a decrease in the amount both low and high

molecular weight DOR (45 kDa and 55 kDa bands) and KOR (45 kDa and 55 kDa

bands). While SIAH2 knock down caused significant increase in the amount of low

molecular weight DOR both species of KOR were increased. As has been demonstrated

with the MOR, the DOR and KOR also exist as differentially glycosylated species. For

the DOR and KOR, the 45 kDa protein has been shown to remain in an intracellular

compartment and is not transported to the cell surface (Chen et al., 2006b; Petaja-Repo et

al., 2000), whereas the 55 kDa protein is the fully mature receptor found at the plasma

membrane (Petaja-Repo et al., 2001). Without performing a double knockdown of

SIAH1 and SIAH2 in these cell lines, there are several interpretations that fit the data

collected from knockdown of SIAH1 or SIAH2 in DOR-HEK and KOR-HEK cells.

These interpretations are discussed in more detail in Chapter 5. One explanation is that

SIAH1 and SIAH2 could possibly have an overlapping role in receptor ubiquitination.

Knockdown of either protein could result in upregulation or overactivation of the other

which would lead to the observed increase in receptor uiquitination. Indeed as with all

cell lines observed knockdown of SIAH1 caused an increase in SIAH2 levels. Another

possibility is that another unknown protein is involved in the ubiquitination of DOR and

KOR and that the SIAH proteins play a role in regulating this unknown protein. Double

knockdowns of both Siah proteins were attempted for both DOR and KOR cell lines, but

102

were unsuccessful. Optimization of the double siRNA transfection in these cell lines may

clarify the role these E3 ligases play in the MOR life cycle.

These interactions between the Siah proteins and the opioid receptors are

surprising considering that opioid receptors are typically downregulated by lysosomal

degradation and not the proteasome (Marchese et al., 2008). However, ubiquitination of

plasma membrane-bound MOR does occur in response to receptor activation and is

required for trafficking events that downregulate receptor ligand binding (Hislop et al.,

2011). This is the first study to address basal (unstimulated) ubiquitination of the MOR.

My results indicate that SIAH1 and SIAH2 may play a role in the ubiquitination the

intracellular form of the MOR under unstimulated conditions. The exact location of the

the low molecular weight, intracellular MOR is not well established, but it is suspected

that some resides within the biosynthetic pathway, possibly in the endoplasmic reticulum

(ER) (Huang and Liu-Chen, 2009). As a glycoprotein, the MOR must be properly folded

and modified within the ER before being translocated to the plasma membrane. If the

MOR misfolds, it is subjected to endoplasmic reticulum-associated degradation (ERAD).

ERAD requires poly-ubiquitination and retro-translocation of the misfolded protein from

the ER lumen to the ER membrane to the cytosol. Once in the cytosol, these proteins are

subjected to degradation via the proteasome (Feldman and van der Goot, 2009).

Interestingly, misfolded delta opioid receptors have been shown to be transported to the

cytoplasmic side of the ER membrane, where they are deglycosylated and conjugated

with ubiquitin before being degraded by the proteasome (Petaja-Repo et al., 2001). It

seems reasonable to assume that the other opioid receptors may be treated similarly. If

Siah proteins are involved in the biosynthesis of MORs, then they may regulate the

103

number and activity of opioid receptors at the cell surface, which is important in the

modulation downstream signaling (Chen et al., 2006b; Petaja-Repo et al., 2000).

Presently, the precise intracellular locations of the Siah proteins are not known.

Evidence continues to mount suggesting that the ubiquitin pathway is involved in

a variety of neuronal activities, including dendritic morphogenesis and synaptic plasticity

which have been implicated in the development of addiction (Robinson and Kolb, 2004;

Tuoc and Stoykova, 2010; Yang et al., 2008). Thus, it will be interesting to determine

how the SIAH proteins regulate the opioid receptors in cells where the both the receptors

and the SIAH proteins are expressed at endogenous levels. Additionally, tissue specific

or conditional knockouts of SIAH1 or SIAH2 could be tested in addiction paradigms to

determine whether this regulation plays a role in the neuronal changes observed in

addiction.

Chapter 4

Effect of Opioid Exposure on MORIPs

4.1 Introduction

The mu-opioid receptor (MOR), which belongs to the Class A family of G protein

coupled receptors (GPCRs), mediates a variety of functions in the body, including

modulation of pain perception, cell proliferation, cell survival, neural development, and

synaptic plasticity (Christie, 2008; Tegeder and Geisslinger, 2004). This receptor couples

preferentially to Gi/o types of G proteins and therefore inhibits adenylyl cyclase activity

and regulates a number of other signaling intermediates such as mitogen activated kinase

(MAPK), phospholipase C (PLC), and various ion channels. The MOR is activated by

endogenous and exogenous opioids, the latter of which include some of the most

effective analgesics, but also the most highly addictive drugs of abuse.

Abuse of drugs or addiction is an abnormal behavior characterized by compulsive,

out-of-control drug use despite negative consequences. The development of these

behavioral abnormalities occurs gradually and progressively during repeated exposure to

the abused drug. In some cases, these abnormalities can persist for a long period of time

after the cessation of use (Nestler, 2004). Thus, drug addiction is seen as a form of drug-

induced plasticity (Russo et al., 2010). Multiple studies have shown that repeated

105

exposure to an addictive drug can alter the types or the levels of genes/proteins expressed

in the brain. In addition, these alterations mediate the function of individual neurons and

related neural circuits which may be responsible for the observed behavioral

abnormalities seen in addicted individuals (Nestler, 2001). Therefore, the identification

of the genes/proteins associated with drug dependence is vital to understanding the

molecular mechanisms underlying addiction.

Many different techniques have been utilized to investigate the gene/protein

expression changes associated with drug exposure and addiction. Proteomic approaches

allow for the study of these changes without the need for a prior hypothesis. Since drug

exposure and addiction involve a large array of interacting proteins, it is well suited for

this type of analysis. However, the lack of a hypothesis, also presents problems. Without

a hypothesis, any found changes have little meaning unless further studies are

implemented. One reason for this is that changes observed in the proteome may not be a

direct consequence of the drug itself, but could represent downstream changes that are

caused by but are far removed from the cellular pathways that are directly affected. For

example, a drug might act directly on the mesolimbic dopaminergic tract. Changes in

proteins in the prefrontal cortex may result as an indirect effect of altered signaling from

the mesolimbic pathway. While this might give you information about the disease state

overall, it provides little evidence as to how the drug itself is working on its receptors.

Another approach is to use a candidate gene(s) approach, where genes/proteins with

known functions or interactions associated with genes or proteins that have already been

shown to be involved in drug exposure or abuse are interrogated for expression changes

during drug exposure or in addiction models.

106

The first proteins shown to interact functionally with the MOR were G proteins,

which modulate canonical G protein-dependent signaling. However, it is now clear that

modulation of MOR signaling and regulation are far more complex (Georgoussi et al.,

2012). Using approaches such as yeast-two hybrid screens, immunoprecipitation,

immunofluorescence, or bioluminescence resonance energy transfer in live cells, it has

been shown that a diverse group of other interacting partners can participate in the

regulation of virtually every aspect of MOR activity. Observations from these studies

have indicated that the MOR can form mulitcomponent protein assemblies, or

signalplexes, involving interactions between themselves, other GPCRs, and with other

interacting proteins. These interactions likely contribute to the process of signaling

downstream of the receptor (Alfaras-Melainis et al., 2009; Georgoussi et al., 2012;

Kabbani et al., 2002; Milligan, 2005).

Few previously identified mu-opioid receptor interacting proteins have been

investigated with regards to altered expression when cells or animals are treated with

opioid drugs. Spinophilin was found to have decreased expression with acute but not

with chronic exposure to morphine in the nucleus accumbens of mice. Additionally,

spinophilin knockout mice exhibited decreased morphine analgesia, but increased

tolerance, physical dependence, and place conditioning to morphine (Charlton et al.,

2008). Acute morphine administration has been demonstrated to cause an increase in

RGS9-2 protein levels in the nucleus accumbens and spinal cord of C57BL/6 mice.

However, mice chronically treated with morphine, showed decreases in RGS9-2 proteins

levels in the same brain regions (Zachariou et al., 2003). These studies suggest that

MORIP expression levels can change with drug exposure and that these changes are

107

likely to have functional consequences, which may contribute to the development of

addiction. Therefore, investigation of MORIP expression upon drug exposure may lead

to the discovery of additional molecular components that may be involved in the

development of the addicted state.

In this chapter, we examine the effect of drug exposure on the SIAH1/MOR

interaction and the expression of selected MORIPs in specific brain regions of morphine

treated mice. We demonstrate that the SIAH1/MOR interaction is altered in response to

morphine and that changes in the expression of several MORIPs may be correlated with

morphine exposure.

4.2 Experimental Procedures

4.2.1 Cell culture and drug treatment

Human neuroblastoma cells (SYH5Y) were cultured in Dulbecco's Modified

Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Human

embryonic kidney (HEK) 293 cells stably transfected with FLAG-tagged mu-opioid

receptor (HEK-MOR), FLAG-tagged delta-opioid receptor (HEK-DOR), or FLAG-

tagged kappa-opioid receptor (HEK-KOR) were maintained in DMEM supplemented

with 10% FBS and 400 μg/mL G418. HEK-MOR and HEK-DOR cell lines were

generously provided by Dr. Mark van Zastrow (University of California San Francisco).

HEK-KOR cells were a gift from Dr. Lee-Yuan Liu-Chen (Temple University School of

108

Medicine). For drug treatment, either 10 μM morphine or 10 μM [D-Ala2, NMe-Phe

4,

Gly-ol5] (DAMGO) was added to cell culture media and cells were incubated for

specified time periods.

4.2.2 Co-immunoprecipitation and Western blot analysis

Human neuroblastoma cells (SHSY-5Y) were exposed to either 10 μM morphine

or 10 μM DAMGO for 30 minutes and 1 hour, then lysed in 1X lysis buffer (20 mM Tris-

HCl pH7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 2.5 mM sodium

pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1% Triton X-100 and

1mg/mL leupeptin) supplemented with protease inhibitor cocktail (Pierce, Rockford, IL).

The MOR was immunoprecipitated from total cell lysates using a polyclonal rabbit anti-

MOR antibody (AB 1580, Millipore, Billerica, MA) coupled to Protein-G Dynabeads

(Invitrogen, Grand Island, NY). Human embryonic kidney cells (HEK) stably transfected

with FLAG-tagged MOR, DOR, or KOR (HEK-MOR, HEK-DOR, HEK-KOR,

respectively) were treated for 1 hour and 21 hours with 10μM morphine. Cells were

collected and lysed in 1X lysis buffer supplemented with protease inhibitor cocktail. The

opioid receptors were immunoprecipitated using polyclonal rabbit anti-FLAG antibody

(Sigma, St. Louis, MO) bound to Protein-G Dynabeads (Invitrogen). Western blot

analysis of immunoprecipitated complexes was performed by using a polyclonal goat

anti-SIAH1 antibody (1:5000 dilution; Abnova, Taipei City, Taiwan) and a bovine anti-

goat HRP-conjugated secondary antibody (1:20,000 dilution; Jackson ImmunoResearch).

Blots were also immunoblotted using Guinea pig anti-MOR antibodies (1:5000 dilution;

109

Neuromics, Ednia, MN) or monoclonal mouse anti-FLAG antibodies (1:5000 dilution;

F1804; Sigma) as a positive control and donkey anti-Guinea pig horseradish peroxidase

(HRP)-conjugated antibodies or goat anti-mouse-HRP conjugated secondary antibodies

(Jackson ImmunoResearch). Mock immunoprecipitations using normal rabbit IgG (Santa

Cruz Biotechnology) were used as negative controls. Immunoreactivity was detected by

ECL Plus.

4.2.3 Animal Care and Drug Treatment

All mouse studies were performed in collaboration with Tom Ferraro (University

of Pennsylvania) in the research laboratories at the Veteran’s Administration Medical

Center (VAMC) in Coatsville, PA, and were approved by the University of Pennsylvania

Institutional Animal Care and Use Committee. C57BL/6J mice were bred in-house,

maintained on a 14hr/10hr light/dark schedule and had food and water available ad

libitum throughout the course of the experiment.

Female mice, 8-9 weeks of age, were implanted sub-cutaneously with a 25 mg

morphine (n=5) or placebo (n=4) pellet. Morphine and placebo pellets were obtained

from the NIDA Drug Supply Program. Mice were anesthetized with ketamine and

zylazine (80 and 10 mg/kg, ip, respectively, then euthanized by cervical dislocation under

carbon dioxide anesthesia four days (96 hr) after pellet implantation. Brains were

harvested and dissected on ice under 5x magnification into 6 regions including prefrontal

cortex, nucleus accumbens, dorsal striatum, midbrain, hippocampus, and cerebellum.

Specimens were frozen on dry ice and stored at -80C.

110

4.2.4 Tissue Preparation and Protein Analysis

Frozen brain tissue obtained from drug or sham treated mice was utilized for protein

expression analysis. Fresh whole-brain tissue from untreated mice was obtained for co-IP

experiments. All tissue was suspended in lysis buffer (50mM Tris-HCl, 1mM EDTA, 150mM

NaCl, 1% NP40, 0.25% deoxycholate, 5mM NaF, 2mM NaVO3) containing protease inhibitors

(cOmplete MINI EDTA free, Roche), homogenized using a microcentrifuge pestle for 2 minutes,

sonicated using a probe sonicator, then centrifuged at 13,000 RPM to remove cellular debris.

For protein expression analysis, samples were analyzed by electrophoresis on 10% SDS-

containing polyacrylamide gels, followed by Western blotting. In addition to antibodies utilized

for co-IP, guinea pig anti-MOR (GP10106, Neuromics, Edina, MN) and Rabbit anti-DOK4

(SAB1300112, Sigma-Aldrich) antibodies were also used to probe brain lysates. For quantitative

analysis of MORIP expression in drug treatment studies, lysates from sham and morphine-treated

mice (20 μg/brain region) were analyzed in quadruplicate. To rule out transfer artifacts, samples

from each brain region were loaded in a different order on each of the four blots. Blots were

scanned using a back-lit scanner and quantitation was performed using IMAGEJ software

(Abramoff et al., 2004). Expression was normalized to total protein (as measured by Ponceau

stain), averaged between replicates, and subjected to a two-tailed Student’s t-test.

111

4.3 Results

4.3.1 Drug exposure alters strength of interaction between SIAH1 and opioid receptors

In an effort to better understand the effect of the MOR/SIAH1 interaction on the

regulation of the MOR under stimulated conditions, I wanted to investigate the effect of

opioid agonists on MOR/SIAH1 complex formation. To determine whether opioid drugs

promote MOR/SIAH1 complex formation, human neuroblastoma cells (SHSY-5Y) were

exposed to either 10 μM DAMGO or 10μM morphine for 30 minutes or 1 hour. These

two drugs were chosen because of their different effects on the trafficking and signaling

of the MOR (Groer et al., 2011). DAMGO causes robust internalization and recycling of

the MOR, while morphine causes markedly slow internalization and no recycling of the

MOR (Christie, 2008). The doses chosen have been shown to saturate receptors and

activate downstream signaling as measured by adenylyl cyclase activity. The treatment

times represent acute exposure that should theoretically capture internalization and

downstream events (such as recycling and ubiquitination), but should not be long enough

to capture degradation of the MOR (Christie, 2008).

To investigate the effects of acute DAMGO and morphine exposure on the

MOR/SIAH1 interaction, SH-SY5Y cells were treated with drug for the designated time

periods. The MOR was immunoprecipitated from equal microgram concentration of total

protein in the lysate (900 μg), resolved by SDS-PAGE, and immunoblotted using goat

anti-SIAH1 antibodies. As seen in figure 4.1A, DAMGO caused an increase in the

amount of SIAH1 that co-immunoprecipitated with the MOR in SH-SY5Y cells after 1

hour of exposure when compared to DMSO treated cells. In contrast, morphine did not

112

cause a change in the amount of co-immunoprecipitated SIAH1 at any time point tested

(Figure 4.1B). These results indicate that DAMGO induced MOR signaling or

internalization enhances the MOR/SIAH1 interaction, which in turn could result in

altered ubiquitination of the receptor.

Figure 4.1: DAMGO treatment of SH-SY5Y cells promotes MOR/SIAH1 complex

formation

(A) SHSY-5Y cells were treated with 10 μM DAMGO for 30 minutes and 1 hour. After

the designated time period cells were lysed and the MOR was immunoprecipitated from

900 μg of total protein using rabbit anti-MOR antibodies bound to Protein G beads.

Eluted proteins were immunoblotted for SIAH1 using goat anti-SIAH1 antibodies. (B)

SHSY-5Y cells were treated with 10 μM morphine for 30 minutes and 1 hour. The MOR

was immunoprecipitated from 900 ug of total protein using rabbit anti-MOR antibodies

bound to Protein G beads. Eluted proteins were immunoblotted for SIAH1 using goat

anti-SIAH1 antibodies. (A and B) Mock IPs using normal rabbit IgG were used as

negative controls. Crude cell lysate (Ly) was used as a positive control and represents

approximately 10% of protein used in IPs.

113

The SHSY cells express both MOR and DOR, which have been shown to

heterodimerize and affect each other’s trafficking and function (Prather et al., 1994). I

therefore wanted to assess opioid drug effects on the interaction of SIAH1 with the

individual opioid receptors in the HEK cell models each expressing a single opioid

receptor subtype. Although, acute morphine exposure did not alter the interaction of

SIAH1 and MOR in SHSY cells, this agonist will act at all three receptors (albeit with

different affinities) and is a known clinically relevant and addictive substance (Neil,

1984). DAMGO is a selective agonist for MOR and would not be useful for the DOR and

KOR studies. HEK-DOR, HEK-KOR, and HEK-MOR cells were exposed to 10 μM of

morphine for 1 hour and 21 hours. For this experiment both acute (1hr) and chronic

effects (21hr) were measured as there were no changed in the MOR/SIAH1 interaction

with acute exposure of morphine in the SHSY cells. Chronic treatment (21hr) has

previously been determined to allow for degradation of these receptors and, therefore

may be a more likely time point to observe a change in the OR/SIAH1 interaction (Afify,

2002; Chen et al., 2006b). Similar to the results with morphine treatment in SHSY-5Y

cells, the MOR/SIAH1 interaction did not change with 1 or 21 hours of treatment in

HEK-MOR cells (Figure 4.2, lanes 11-13). The DOR/SIAH1 interaction, increased with

both acute and chronic morphine treatment (Figure 4.2, lanes 3-5), whereas the

KOR/SIAH1 interaction did not change. These results indicate that acute morphine

exposure may only alter the interaction of SIAH1 with the DOR. However, the IP lanes

appear to have a double band for SIAH1 when immunoprecipitated with DOR and KOR.

This double band could be due to a number of things, such as degradation of SIAH1 or

posttranslational modification. Taken together these results suggest that morphine may

114

have a selective effect on the DOR/SIAH1 interaction by promoting the formation of this

complex.

Figure 4.2: Morphine treatment promotes DOR/SIAH1 complex formation

(A) HEK-DOR, HEK-KOR, and HEK-MOR cells were treated with 10 μM morphine for

1 hour and 21 hours. After the designated time period cells were lysed and the DOR,

KOR, or MOR was immunoprecipitated using rabbit anti-FLAG antibodies bound to

Protein G beads. Eluted proteins were immunoblotted for SIAH1 using goat anti-SIAH1

antibodies. Mock IPs using normal rabbit IgG were used as negative controls. Crude cell

lysate (Ly) was used as an additional positive control and represents approximately 5% of

protein used in IPs.

4.3.2 Interaction of MOR and MORIPs in Rodent Brain

To determine whether the interaction between the MOR and MORIPS may occur

in vivo, we analyzed the expression of MOR and several MORIPS in various mouse brain

regions. Not all commercially available antibodies were reliable when used to detect

MORIPs in the brain. For example, antibodies available for SIAH2, zyxin, and DOK5

were not able to detect proteins of the proper size in brain lysates. Other antibodies, such

as AUP1 could detect the protein in cell lysates, but yielded many non-specific bands in

brain lysates. Therefore, only MORIPs whose commercially available antibodies

detected a protein of the correct molecular weight in brain lysates were chosen for further

115

study. As shown in Fig. 4.3A, the MOR and its interacting partners DOK4, SIAH1,

VAPA, and WLS were each expressed in cerebellum, hippocampus, nucleus accumbens,

midbrain, prefrontal cortex, and dorsal striatum. Co-expression of the MOR and each of

the MORIPs in all brain regions tested is consistent with the idea that the MOR and its

binding partners have the potential to interact in multiple brain regions. To verify

interaction, the MOR was immunoprecipitated from a whole mouse brain lysate, and

immunocomplexes probed for the presence of several MORIPs. As shown in Fig. 4.3B,

SIAH1, VAPA, and WLS were able to co-IP with MOR but not with rabbit-IgG alone,

suggesting that these MORIPs are capable of interacting with MOR in mouse brain.

DOK4 was not tested due to crossreactivity of the antibody with rabbit IGG used for

immunoprecipitations.

116

Figure 4.3: MORIP expression and interaction with MOR in mouse brain

(A) Expression of MOR and MORIPS in mouse brain regions. Western blots containing

lysates prepared from mouse cerebellum (C), hippocampus (H), midbrain (M), nucleus

accumbens (N), prefrontal cortex (P), and striatum (S) were probed with Guinea pig anti-

MOR, rabbit anti-DOK4, goat anti-SIAH1, mouse anti-VAPA, and chicken anti-WLS

antibodies. (B) Co-immunoprecipitation. The MOR was immunoprecipitated from

whole mouse brain lysates using rabbit anti-MOR antibodies. Immunocomplexes were

probed for the presence of SIAH1, VAPA, or WLS using MORIP-specific antibodies.

Lysate lanes (L) contain 5% of the total protein prepared from whole mouse brain lysate

compared to the mock (M, rabbit IGG) and immunoprecipitation (IP) lanes. Experiments

performed by Jessica Petko at The Pennsylvania State University College of Medicine.

117

4.3.3 The Effect of Morphine on MORIP Expression

To determine whether chronic morphine exposure alters the protein levels of

MORIPs, we performed Western blot analysis of selected MORIPs (DOK4, SIAH1, and

WLS) in brain regions thought to be involved in response to opioid drug exposure. MOR

levels are not reported for this experiment, as the antibody lot utilized resulted in high

background and very light bands that were unable to be quantified. Mice were implanted

subcutaneously with either saline (sham n=4) or morphine (n=5) pellets for 96 hours,

then euthanized, and the brains dissected.

As shown in Figure 4.4, no significant changes were detected in any of the

MORIPs tested in either cerebellum or nucleus accumbens. However, in response to

morphine exposure, DOK4 levels were decreased by ~50% in hippocampus (p=0.02) and

midbrain (p=0.003). In morphine-treated animals, SIAH1 expression was reduced by

48% in hippocampus (p=0.008) but showed a 55% increase in midbrain (p=0.03) (Figure

4.4). WLS levels were decreased by 49% in midbrain (p=0.006) and 39% in striatum

(p=0.02) following drug exposure (Figure 4.4). Although every effort was made to

ensure equal loading of proteins, some samples consistently contained higher amounts of

total protein than others (e.g., sham versus morphine treated samples in hippocampus).

Despite these differences in protein loading, our data suggest that morphine treatment

may lead to significant alterations in MORIP protein expression in various regions of

mouse brain.

118

Figure 4.4: MORIP expression in brain regions of morphine treated mice

Mice were treated for 96 hours with either morphine-containing (n=5) or placebo (n=4)

pellets. Animals were sacrificed, brain regions dissected, and Western blots of selected

MORIPs probed with MORIP-specific antibodies. Each panel contains a representative

blot for a MORIP in the specified brain region (n=4 blots/MORIP/brain region). Total

protein was quantitated by Ponceau stain of the blot prior to antibody probing. Bar graphs

represent the average pixel density (as determined by IMAGEJ) of four blots for each

brain region normalized to total protein and placebo treatment. Data was analyzed using

a two-tailed Student's t-test. Error is expressed as standard error of the mean. * indicates

a statistically significant difference (p<.05) between sham and morphine treatment.

Experiments performed by Jessica Petko at The Pennsylvania State University College of

Medicine.

119

4.4 Discussion

My results indicate that the strength of the interaction between SIAH1 and the MOR

changes with acute exposure to DAMGO, but not with acute or chronic morphine treatment. In

line with this finding, DAMGO has been shown to cause ubiquitination of mu opioid receptors

that peaks at 1 hour of agonist stimulation (Groer et al., 2011). An acute treatment with

DAMGO causes robust internalization of the MOR within 30 minutes which may make MOR

available in the cytosol for an interaction with SIAH1. In contrast, morphine causes slow

internalization and no ubiquitination of the MOR which could explain why no increase in

SIAH1/MOR interaction was detected. Alternatively, the differences seen in MOR/SIAH1

complex formation could be due to proteins that are brought to the signaling complex during

activation of the MOR. For example, DAMGO stimulation of MOR brings β-arrestin 2 to the

MOR signaling complex, but morphine does not. Additionally, loss of β-arrestin 2 prevents the

DAMGO-stimulated ubiquitination of the MOR (Groer et al., 2011). Degradation of the MOR in

response to agonist (DAMGO or morphine) requires several hours of agonist binding (Afify,

2002; Chen et al., 2006b). Although DAMGO was not studied under chronic conditions, no

change in the MOR/SIAH1 interaction was detected with morphine. Together, these results may

indicate that SIAH1 may be involved in agonist stimulated ubiquitination (by DAMGO) of the

MOR as well as unstimulated ubiquitination (chapter 3).

Morphine was used to determine the effects of acute and chronic receptor activation on

the interaction of the individual opioid receptors and SIAH1. With chronic morphine treatment,

the interaction between SIAH1 and DOR increased while the KOR/SIAH1 interaction did not

change. These results are interesting as morphine has a lower binding affinity for DOR and

120

KOR and likewise only weakly activates these receptors (Keith et al., 1996; Trescot et al., 2008).

While DOR and KOR can be ubiquitinated in response to selective agonists, nothing is known

about the ubiquitination these receptors in regards to morphine stimulation (Henry et al., 2011;

Li et al., 2008). Morphine does not cause ubiquitination of MOR, but this does not rule out this

possibility for DOR or KOR. In addition, little is known about the trafficking of these receptors

in response to morphine exposure. Therefore, we cannot make any conclusions as to whether

trafficking events are changing the DOR or KOR interaction with SIAH1.

The study of MORIP levels in morphine treated mice, utilized the inbred strain

C57BL/6J. This strain has been found to be more sensitive to the rewarding effects of morphine

than other strains, such as DBA/2J, in self-administration and conditioned place preference

paradigms (Elmer et al., 2010). However, our study did not use a reward paradigm (self-

administration or conditioned place preference), but was designed to detect differences between

those individuals exposed to morphine and naïve individuals. Although behavior was not

measured in this study, 96 hour morphine treatment is known to lead to changes in dendritic

morphology and neurogenesis associated with synaptic plasticity and addiction (Eisch et al.,

2000). Additionally, these changes are found to occur, regardless of the method of

administration (pellet, experimenter, or self-administration) (Robinson and Kolb, 2004).

Therefore this kind of study can be a useful tool when combined with behavioral paradigms to

tease out which changes are due to the effect of the drug and which may be due to behavior.

An inbred strain of mouse was utilized in this analysis, because it should control for

genetic variability between animals as they should be homozygous at almost all loci. Even with

this homogeneity of mice, there are some differences in the level of MORIPs detected within

treatment groups. The explanation for this interanimal variability is unknown. However, it may

121

result from genetic variation, such as single nucleotide polymorphisms (SNPs) between animals.

According to the Mouse Genome Informatics database, the C57BL/6J strain can have SNPs in

SIAH1 (Siah1a of mouse), DOK4, and WLS. If these SNPs are found in some individuals and

not others in this study, it may help to explain the observed variation in the level of expression of

a particular MORIP in a mouse. However, no studies have investigated the role of single

nucleotide polymorphisms or splice variants in SIAH1, DOK4, or WLS in regards to the

expression of these proteins.

Knockout mouse experiments have shown that MOR is the primary mediator of the

analgesic and rewarding properties of opioid drugs. Since MORIPs regulate the functional

output of the MOR, they represent potential candidates for proteins that may contribute to the

underlying molecular mechanisms leading to drug addiction. Protein expression analysis of

selected brain regions from morphine-treated mice indicate that the expression of DOK4, SIAH1,

and WLS are altered in distinct brain regions following chronic morphine exposure. Morphine

has been shown to decrease WNT secretion in a cell culture system, and that WLS shifts from a

predominantly cytosolic to a more plasma membrane-associated distribution in cultured cells as

well as rat striatal neurons after morphine treatment (Jin et al., 2010a; Reyes et al., 2012). The

decrease in WLS expression observed in midbrain and striatum of mice after morphine

administration could possibly lead to long term decreases in WNT secretion within the CNS. It

is possible that decreased Wnt secretion could be responsible for many of the observed effects of

chronic opioid exposure including decreased neurogenesis and decreased dendritic spine density

that are known to be affected by WNT signaling (Eisch et al., 2000; Liao et al., 2005).

The implications of decreased SIAH1 expression in the hippocampus and its increased

expression in the midbrain could be enhanced by looking at MOR levels. As mentioned in the

122

results this was attempted, but changes in antibody performance (change of Lot number) did not

allow the completion of this analysis. Although the functional relevance of changes in DOK4

and SIAH1 expression after morphine exposure is currently unknown, it is of interest that these

changes in protein expression were observed within specific brain regions, rather than

throughout the entire brain. Thus the changes in protein expression we detected may play an

important role in the response to opioid drug exposure mediated by these brain regions. In the

future, it should become possible to examine the role of these MORIPs in behavioral aspects of

opioid addiction using animal models of opioid self-administration.

In summary the findings presented in this chapter indicate that while the non-

internalizing agonist morphine does not affect the MOR/Siah1 interaction, the internalizing

agonist DAMGO enhances this interaction. This indicates that although the SIAH proteins

appear to be involved in a basal level of ubiquitination and receptor degradation (as described in

chapter 3), there may also be a role for SIAH1 in agonist stimulated MOR regulation. In

addition, several newly identified MORIPS including SIAH1 are co-expressed with and interact

with MOR in the mouse brain. Finally, several MORIPs display altered expression in the mouse

brain during chronic exposure to morphine. This finding not only suggests a role for these

proteins in the drug adaptation process, but also validates the interaction screen/candidate gene

approach to identifying potential players in development of addiction.

Chapter 5

Overall Discussion

The overall aim of this thesis was to characterize novel mu opioid receptor

interacting proteins (MORIPs) in an attempt to provide new understandings into the

cellular and molecular mechanisms that underlie the development of opioid addiction.

This goal was founded upon several lines of evidence, including the central role of the

MOR in mediating both the analgesic and rewarding effects of opioid agonists (Matthes

et al., 1996), the growing body of evidence suggesting that G protein-coupled receptors

(GPCRs) are part of large signaling complexes whose protein constituents have not all

been identified (Kabbani and Levenson, 2007), and that we still do not have a clear

understanding of the basic biological underpinnings of this disease. In working towards

this aim, several novel MORIPs were discovered, the function of the MOR/SIAH1/2

interaction was explored, and changes in MORIP protein levels were detected in

morphine treated mice.

Possible roles of Novel MORIPs in morphine exposure

Utilization of traditional and modified yeast two-hybrid (Y2H) screens identified

10 novel and two previously discovered components of the MOR signaling complex.

Some of these MORIPs were shown to interact with other opioid receptors, which

124

suggests that they may be universal regulators of opioid function. The conventional Y2H

was unique as it utilized the second intracellular loop of the MOR, which had not

previously been studied in terms of interacting proteins. Several of the novel MORIPs

identified in these screens have been shown to share common cellular functions that may

correlate with changes that have been observed in the development of addiction, such as

synaptic plasticity and neurogenesis. A summary of the cellular function of MORIPs

found in this study and in previous studies is depicted in Figure 5.1.

Figure 5.1: MOR interaction map

Web of newly identified and previously known MOR interacting proteins. MORIPs were

grouped based on previously characterized functional properties. MORIPs identified in

the current study are depicted by green shaded circles, while previously identified

MORIPs are represented by white circles. Many of the newly identified MORIPs also

interact with DOR and KOR (see Chapter 2 Results).

125

Since MORIPs have the potential to regulate the functional output of the MOR, they

represent potential candidates for proteins that may contribute to the underlying molecular

mechanisms leading to drug addiction. Protein expression analysis of selected brain regions

from morphine-treated mice indicate that the expression of DOK4, SIAH1, and WLS are altered

in distinct brain regions following chronic morphine exposure. Morphine has been shown to

decrease WNT secretion in a cell culture system, and WLS shifts from a predominantly cytosolic

to a more plasma membrane-associated distribution in cultured cells as well as rat striatal

neurons after morphine treatment (Jin et al., 2010a; Reyes et al., 2012). The decrease in WLS

expression observed in midbrain and striatum of mice after morphine administration could

possibly lead to long term decreases in WNT secretion within the CNS. The midbrain contains

the ventral tegmental area (VTA) nuclei that are the main sources of dopamine (DA) in the brain,

whereas other regions, such as the nucleus accumbens, striatum, and prefrontal cortex are the

target regions for DA released from the VTA. It is widely accepted that addictive drugs cause

functional changes these brain regions. These functional changes may manifest as changes in

neuron firing, strengthening or weakening of synapses, changes in neuron morphology, and

neurogenesis (Nestler, 2004; Nugent et al., 2007; Robinson and Kolb, 2004; Russo et al., 2010).

Coincidentally, dendritic spine density and neurogenesis are known to be affected by WNT

signaling (Eisch et al., 2000; Liao et al., 2005). Therefore, it has been hypothesized that

decreased WNT secretion could be responsible for many of the observed structural and

functional effects of chronic opioid exposure (Jin et al., 2010).

In the nervous system, DOK4 has been shown to be required for axon myelination

(Blugeon et al., 2011) and the regulation of growth derived neurotropic factor (GDNF)-

dependent neurite outgrowth (through activation of the Rap1-ERK1/2 pathway) (Uchida et al.,

126

2006). Neurite outgrowth has been proposed as a possible mechanism for plasticity in the

hippocampus (Ring et al., 2006). A hypothetical explanation for the decreased levels of DOK4

in the hippocampus is that the reduction in DOK4 protein may contribute to reduced neurite

outgrowth and consequently reduced plasticity in this brain region. Determining the function of

the MOR/DOK4 interaction in relation to neuronal morphology, MAPK-mediated signaling, and

other processes associated with MOR activation and opioid addiction may clarify the

consequences of reduced DOK4 in response to morphine exposure in mice.

The expression of SIAH1 mRNA has been found to be decreased in the middle temporal

gyrus (includes the hippocampus) of patients with a history of alcohol abuse (Sokolov et al.,

2003). Our study found a decrease in SIAH1 protein levels in the hippocampus of morphine

treated mice. The method of morphine treatment performed in this study has previously been

shown by an independent group to cause structurally changes in the hippocampus that are

associated with the development of addiction (Liao et al., 2005; Nestler, 2004). Additionally,

other proteins involved in the ubiquitin proteasome pathway have been shown to be altered in

addiction (Bingol and Sheng, 2011; Tuoc and Stoykova, 2010). Based on the results from

Chapter 3, it is tempting to speculate that SIAH levels play an important role in the addiction

process by direct regulation of MOR ubiquitination, trafficking, and/or degradation. Further

characterization of the roles of SIAH E3 ligases in ligand-mediated MOR regulation will be

crucial to determining whether this speculation may be possible.

Although the functional relevance of changes in WLS, DOK4 and SIAH1 expression

after morphine exposure is currently unknown, it is of interest that these changes in protein

expression were observed within specific brain regions, rather than throughout the entire brain.

Thus, the changes in protein expression we detected may play an important role in the response

127

to opioid drug exposure mediated by these brain regions. In the future, it should become

possible to examine the role of these MORIPs in behavioral aspects of opioid addiction using

animal models of opioid self-administration. Knockout animals of WLS, SIAH1, or DOK4

could be used in self-administration paradigms to determine the possible role that they play in

the addiction process. This type of approach allows for the measurement of drug seeking, taking,

and reinstatement. The affect on these addiction-like behaviors due to the loss of a particular

MORIP, has already been used for spinophilin (a previously identified MORIP). The loss of

spinophilin in mice resulted in a greater sensitivity to the rewarding properties of morphine and

the development of a higher degree of physical dependence (Charlton et al., 2008). The use of a

similar approach for WLS, SIAH1, and DOK4 may be able to clarify the roles of these proteins

in behavior. Additionally, the role of degradation could be explored through the use of

lysosomal and proteasomal inhibitors in conjunction with a self-administration paradigm with

knockout animals.

The DOR/SIAH1/2 and KOR/SIAH1/2 interactions

The MOR is known to form functional dimers with the other two major opioid

receptors (Chakrabarti et al., 2010; Gomes et al., 2000), and all three of these receptors

have been demonstrated to interact with both SIAH1 and SIAH2 (Chapter 2). Thus, it

was important to investigate the role of the Siah proteins in the regulation of the DOR

and KOR. MOR will be discussed in more detail; therefore, DOR and KOR will be

discussed first. In chapter 3, SIAH1 and SIAH2 were knocked down in HEK-DOR and

HEK-KOR cells. The results from those experiments yield several possible models for

128

the function of the Siah proteins in the basal (unstimulated) regulation of DOR and KOR.

Two of these models will be discussed below.

In the first model, both SIAH1 and SIAH2 are capable of conjugating ubiquitin to

the DOR and the KOR (Figure 5.2A). In other words, the Siah proteins may compensate

for the loss of the other. This ubiquitination of DOR and KOR by the Siah proteins leads

to different fates for the receptors. Ubiquitination by SIAH2 appears to signal for

degradation of these receptors, as knockdown of SIAH2 caused an increase in the protein

levels of both DOR and KOR. In contrast, ubiquitination by SIAH1 does not appear to

signal for degradation, as knockdown of SIAH1 caused a decrease in the protein levels of

both DOR and KOR. In the second model, the Siah proteins regulate the ability of

another E3 ligase to conjugate ubiquitin to DOR and KOR. SIAH 1 may regulate

SIAH2, SIAH2 may regulate SIAH1, or the regulation may require a heterodimers of

SIAH1/SIAH2, which regulate a third E3 ligase. This E3 ligase would then conjugate the

DOR or KOR with ubiquitin and signal for degradation or trafficking (Figure 5.2B). This

model predicts increased ubiquitination with SIAH1 or SIAH2 knockdown. Consistent

with this model, knockdown of either SIAH1 or SIAH2 resulted in increased receptor

ubiquitination. Without the benefit of double knockdown experiments in HEK-DOR and

HEK-KOR cells, it is difficult to discern which model may best represent what is

occurring in these cells with regards to DOR and KOR regulation.

The experiments performed in this dissertation did not look at the effects of

opioid agonist stimulation in combination with SIAH1 or SIAH2 knockdown. However,

the effect of acute and chronic morphine treatment on DOR/SIAH1 and KOR/SIAH1

complex formation was examined. Although morphine weakly binds and activates DOR

129

and KOR, chronic morphine treatment resulted in an increase in the formation of

DOR/SIAH1 complexes. Therefore, it is possible that SIAH1 may play an additional role

in the regulation of DOR and/or KOR stimulated with agonist. Experiments utilizing

DOR- and KOR-specific agonists in combination with knockdown of SIAH1 and SIAH2

may help clarify the roles of the Siah proteins in receptor regulation.

Lastly, it will be important to determine the functional consequences of the

ubiquitination of these receptors both basally and upon activation. My results suggest that

ubiquitination in the absence of SIAH1 allows for receptor degradation, whereas the

absence of SIAH2 does not. Since both the lysosomal and proteasomal pathways can

degrade the DOR and KOR, it will be important to determine which pathway is being

used (Chaturvedi et al., 2001; Henry et al., 2011; Li et al., 2008). This could be

accomplished by blocking the lysosome and the proteasome with inhibitors in

combination with the knockdown of SIAH1 and SIAH2.

130

Figure 5.2: Models for SIAH1 and SIAH2 function in HEK-DOR and HEK-MOR

cells

(A) SIAH1 and SIAH2 are capable of ligating ubiquitin to the DOR and/or KOR. This

ubiquitination signal has three possible consequences, degradation by the lysosome,

degradation by the proteasome, or a trafficking event. (B) SIAH1 and SIAH2 or a

SIAH1/SIAH2 heterodimer may regulate another E3 ligase, which acts to ubiquitinate

DOR and/or KOR. This ubiquitination signal has three possible consequences,

degradation by the lysosome, degradation by the proteasome, or trafficking of the

receptor(s).

Model for the Interaction between the MOR and the Siah proteins

The discovery that SIAH1 and SIAH2 interact with the MOR was surprising,

considering that the MOR is usually downregulated by lysosomal degradation and not via

the proteasome. However, ubiquitination of the MOR does occur and has been shown to

be required for trafficking events that downregulate receptor ligand binding after agonist

131

stimulation (Hislop et al., 2011). Additionally, this ubiquitination has been shown to be

ligand dependent, as DAMGO but not morphine, will cause ubiquitination of the MOR

(Groer et al., 2011).

The work presented in this dissertation suggests a previously unrecognized role

for ubiquitination in the regulation of unstimulated MOR expression via interaction with

SIAH1 and SIAH2. The combined overexpression data and Siah proteins knockdowns,

yield several possible models for their role in MOR regulation. In both models, SIAH1

acts to regulate the levels of SIAH2 protein, either directly (Figure 5.3A) or indirectly

(Figure 5.3B). This is consistent with the observation that Siah2 proteins levels increased

with SIAH1 knockdown in all cell lines tested. Both models also suggest that SIAH2 is

an E3 ligase for the MOR, as SIAH2 knockdown resulted in a decrease in receptor

ubiquitination. However, SIAH2 knockdown did not result in increased levels of MOR

protein, suggesting that SIAH2-mediated ubiquitination of the MOR alone is not

sufficient for degradation of the receptor. It is possible that another protein is required to

escort the ubiquitinated MOR to the site of degradation (lysosome or proteasome). It is

tempting to speculate that this shuttle protein may be another novel MORIP, such as

AUP1 or CSN5. The effect of the knockdown of these proteins on ubiquitination and

receptor protein levels has not yet been investigated and may shed some light on the role

of these ubiquitin pathway proteins in the MOR life cycle. Another alternative is that

SIAH1 may serve a dual role as the SIAH2 regulator and a shuttle protein. The double

knockdown of both SIAH1 and SIAH2 yielded results that suggest that both SIAH1 and

SIAH2 are required for the ubiquitination of the MOR.

132

Figure 5.3: Simplified model of Siah protein-mediated MOR degradation

(A) SIAH1 inhibits SIAH2, which ubiquitinates MOR. Degradation via either the

lysosomal or proteasomal pathway requires a shuttle protein to bring the ubiquitinated

MOR to the site of degradation. (B) SIAH1 may inhibit another protein that is

responsible for activation of SIAH2, which ubiquitinates the receptor. Either the

lysosomal or proteasomal degradation requires a shuttle protein to bring the ubiquitinated

MOR to the site of degradation.

Treatment of SH-SY5Y cells with DAMGO and morphine, showed different

effects on the SIAH1/MOR interaction. DAMGO has been shown to cause rapid

internalization, recycling, and ubiquitination of the MOR, while morphine treatment

results in much slower internalization and no recycling or ubiquitination of receptors

(Christie, 2008; Hislop et al., 2011). The interaction between SIAH1 and the MOR was

strengthened with acute DAMGO treatment, but did not change with either acute or

chronic morphine treatment. This observation indicates that DAMGO, but not morphine

promotes the formation of MOR/SIAH1 complexes. One hypothesis is that the increased

133

interaction detected between MOR and SIAH1 with DAMGO stimulation, results in the

ubiquitination of the MOR. In contrast to the role of SIAH1 in unstimulated MOR

regulation, DAMGO stimulated MOR regulation suggests that SIAH1 may be the

putative E3 ligase responsible for the ubiquitination of the MOR observed by Groer and

colleagues (2011). Unfortunately, probing the Western blots from these experiments

with anti-ubiquitin antibodies was unsuccessful. An alternative explanation is that

DAMGO treatment increases SIAH2 activity resulting in MOR ubiquitination and that

the ubiquitination signals SIAH1 to form an interaction with the MOR. Determining the

affect of DAMGO treatment on the MOR/SIAH2 interaction was attempted, but was

unsuccessful. Unfortunately re-probing the Western blots from these experiments with

the anti-SIAH2 antibody did not produce bands. In the future it will be important to

determine the effect of DAMGO and morphine treatment on the MOR/SIAH2

interaction. In addition, acute and chronic DAMGO treatment of cells in which the Siah

proteins have been knocked down may clarify the roles of the Siah proteins in regulation

of agonist stimulated MOR.

Interestingly, not all MOR species were sensitive to the effects of SIAH1, as only

the low molecular weight species (50 kDa band) significantly changed. This low

molecular weight, intracellular species has been postulated to represent MOR in the

biosynthetic pathway (Huang and Liu-Chen, 2009). Therefore, it is possible that some of

this MOR species is associated with the endoplasmic reticulum or another intracellular

compartment. Proteins destined for insertion into the plasma membrane enter the

endoplasmic reticulum (ER) in an unfolded state and generally leave after they have

reached their native states. However, folding in the ER is often slow and inefficient and a

134

substantial fraction of polypeptides fail to reach their native state. The cell must

continuously scrutinize these newly synthesized proteins and remove those that are

terminally misfolded (Smith et al., 2011). High fidelity of the ER quality control is

crucial for the functionally integrity of the cell. To illustrate this point, even subtle

mutations that would not dramatically affect protein function can lead to retention of the

mutant protein within the ER, thereby preventing its physiologically relevant activity.

This has been shown for a variety of proteins, including cystic fibrosis transmembrane

conductance regulator (CFTR), V2-vasopressin receptor, and many GPCRs, including the

MOR (Chaipatikul et al., 2003). However, retention and degradation of incorrectly

folded proteins may not be restricted to products of mutated genes. It has been proposed

that as much as 30% of newly synthesized proteins in various cell types never reach their

correct native structure (Schubert et al., 2000). In fact, it has been demonstrated that only

40% of newly synthesized delta opioid receptors are transported to the cell surface in

human embryonic kidney cells (Petaja-Repo et al., 2000). The number and activity of

opioid receptors on the cell surface are important factors in determining the capacity of

these receptors to modulate downstream signaling molecules (Law et al., 2000).

The involvement of the Siah proteins as potential E3 ligases involved in ERAD is

one explanation for the interaction between SIAH1 and 2 and MOR under non-stimulated

conditions. A number of ERAD ligases have been discovered in mammalian cells. Most

of them, like RNF5/RMA1, TEB4, RFP2, Kf-1, and TRC8 are poorly characterized with

only a few targets identified for each of them. Those that have been investigated in

greater detail, such as Hrd1 (synoviolin) and gp78 (autocrine motility factor, AMFR)

have numerous single and overlapping substrates (Mehnert et al., 2010). The majority of

135

these ERAD ligases have a transmembrane domain which tethers them to the ER

membrane. Analysis of the Siah proteins using PRED-TMR, a program designed to

predict transmembrane domains, did not detect any putative transmembrane domains

(Pasquier et al., 1999). In fewer cases, cytosolic ligases, such as Parkin, have been

demonstrated to promote turnover of selected proteins via ERAD (Imai et al., 2002;

Yoshida et al., 2002). Thus, the Siah proteins may be additional cytosolic E3 ligases that

participate in the ERAD pathway. Determining the precise location where the interaction

between the Siah proteins and the MOR occurs could help determine whether or not this

interaction may play a role in the ER-associated degradation of the MOR. This could be

tested using immunogold labeling of MOR and Siah proteins and electromicrography.

Currently, there is a lack of data on the expression profile of SIAH1 and SIAH2

and the contribution of these E3 ligases to ERAD. The criteria that drive individual

polypeptides into distinct ERAD pathways also remain unknown (Kostova et al., 2007;

Mehnert et al., 2010). However, one could hypothesize that the activity of individual

ligases or their cofactors may be controlled spatially, temporally, or in a tissue-specific

manner to allow cells to adapt to varying physiological conditions. In addition, the

amount of some of these ligases and cofactors may be regulated by the proteolysis of

other ERAD ligases (Mehnert et al., 2010). In fact, the ERAD ligase Hrd1 has been

shown to promote gp78 ubiquitination and degradation under certain conditions, thereby

affecting the degradation of gp78 target proteins (Shmueli et al., 2009). Thus, the

regulation of SIAH2 by SIAH1 may be important in preventing the targets of SIAH2

from ubiquitination under certain conditions, such as basal MOR turnover.

136

In the model of MOR degradation via ERAD, misfolded MOR would be

recognized by ER chaperones, which would bring it to an ER-resident E3 ligase for

ubiquitination (Figure 5.4A). Once ubiquitinated, the misfolded MOR would be

translocated through a channel protein to the cytosol. Cytosolic de-ubiquitinating

enzymes (DUBs) would then remove the ubiquitin from the MOR (Figure 5.4B).

Cytosolic chaperones would bind the misfolded MOR and SIAH2 would ubiquitinate the

MOR to signal for degradation (Figure 5.4C). The SIAH2-mediated ubiquitination of

MOR, would facilitate the shuttling of the misfolded protein to the proteasome by SIAH1

(Figure 5.4D). However, further evidence is needed to support or refute this model. One

method of determining the involvement of an E3 ligase in ERAD, is to induce ER stress

in cells. Sustained ER stress eventually leads to a stress response, unfolded protein

response (UPR). In an attempt to relieve this stress, cells will increase the synthesis of

ER-resident chaperones and other proteins involved in ER-associated protein

degradation. Therefore, if the Siah proteins are involved in ERAD, exposing cells to an

N-linked glycosylation inhibitor, such as tunicamycin, may result in increased expression

of the Siah proteins.

137

Figure 5.4: Model of the role of SIAH1 and SIAH2 in ER-associated degradation of

the MOR

(A) MOR (black line) is recognized by ER chaperones and is partially translocated

through a protein conducting channel complex/retrotanslocon (brown). MOR is

conjugated with chains of ubiquitin (gray circles) by an ER-resident ubiquitin ligase (E3)

and its cognate ubiquitin conjugating enzyme (E2) on the cytosolic face of the ER

membrane. (B) The ubiquitin chains on the MOR serve as an exit signal and MOR moves

out of the ER. This movement is facilitated by the removal of ubiquitin molecules by de-

ubiquitinating enzymes (DUB). (C) The dislocated MOR is ubiquitinated a second time

by SIAH2. (D) SIAH1 recognizes the ubiquitnated MOR and shuttles it to the

proteasome for degradation. Adapted from (Tsai and Weissman, 2011).

Taken together my results suggest that the roles for the Siah proteins in

ubiquitination of the opioid receptors are different. The majority of my results suggest

SIAH2 is a putative E3 ligase for all three of the opioid receptors. Testing the ability of

SIAH2 to conjugate ubiquitin to the individual opioid receptors in in vitro ubiquitination

assays would confirm or refute SIAH2 as an E3 ligase for the opioid receptors. In

contrast, SIAH1 does not appear to be a putative E3 ligase for the unstimulated MOR, but

instead may be a shuttle protein or chaperone that escorts the MOR. However, my

results do not eliminate SIAH1 as a putative E3 ligase for DOR and KOR.

138

The precise role that ubiquitination by SIAH1 or SIAH2 may be playing in the

life cycle of the opioid receptors is still unknown. For the MOR, ubiquitination by

SIAH2 appears to eventually lead to receptor degradation. In the future it will be

important to determine whether SIAH2- mediated ubiquitination results in lysosomal or

proteasomal degradation of the MOR. For the DOR and KOR, the role of SIAH1 and/or

SIAH2 ubiquitination is much less clear. Ubiquitination of DOR and KOR may also lead

to degradation, but it may also result in trafficking of these receptors. Tracking the

intracellular location of opioid receptors under both unstimulated and agonist-stimulated

conditions may shed some light on a more precise role for ubiquitination in the life cycle

of these receptors.

In summary, utilization of traditional and modified yeast two-hybrid (Y2H)

methods identified ten novel and two previously reported MORIPs. Directed Y2H and

GST-PD studies were used to confirm interaction and map the binding site for several

MORIPs to the second intracellular loop of MOR. Many MORIPs were demonstrated to

interact with full length MOR, as well as the DOR and KOR in the context of cultured

human cells. Additionally, chronic morphine treatment was shown to affect the protein

levels of WLS, SIAH1, and DOK4 in specific brain regions of mice. Functional

characterization of two of these novel MORIPs, SIAH1 and SIAH2, has suggested

distinct roles for these proteins in the basal (unstimulated) regulation of the MOR.

SIAH2 is a putative E3 ligase for the MOR, whereas SIAH1 appears to play a role in the

trafficking of the MOR to the site of degradation. The Siah proteins also may

ubiquitinate and degrade or traffic the DOR and KOR. Identification and

characterization of the full panorama of MORIPs represents a critical step in

139

understanding the normal regulation of the receptor life-cycle, as well as how receptor

signaling or trafficking may be hijacked in response to drugs of abuse. The MORIPs

identified in this and other studies may represent key players involved in other opioid-

mediated processes such as neural development, nociception, aversion, and synaptic

plasticity. As the list of MORIPs grows, and their contributions to receptor function

become better understood, it is possible that some of these proteins will also become

targets for new drug development to prevent and/or treat opioid addiction.

REFERENCES

Abbadie, C., Gultekin, S.H., and Pasternak, G.W. (2000a). Immunohistochemical

localization of the carboxy terminus of the novel mu opioid receptor splice variant MOR-

1C within the human spinal cord. Neuroreport 11, 1953-1957.

Abbadie, C., Pan, Y., Drake, C.T., and Pasternak, G.W. (2000b). Comparative

immunohistochemical distributions of carboxy terminus epitopes from the mu-opioid

receptor splice variants MOR-1D, MOR-1 and MOR-1C in the mouse and rat CNS.

Neuroscience 100, 141-153.

Abbadie, C., Pan, Y.X., and Pasternak, G.W. (2000c). Differential distribution in rat

brain of mu opioid receptor carboxy terminal splice variants MOR-1C-like and MOR-1-

like immunoreactivity: evidence for region-specific processing. J Comp Neurol 419, 244-

256.

Abbadie, C., Pan, Y.X., and Pasternak, G.W. (2004). Immunohistochemical study of the

expression of exon11-containing mu opioid receptor variants in mouse brain.

Neuroscience 127, 419-430.

Abbadie, C., Pasternak, G.W., and Aicher, S.A. (2001). Presynaptic localization of the

carboxy-terminus epitopes of the mu opioid receptor splice variants MOR-1C and MOR-

1D in the superficial laminae of the rat spinal cord. Neuroscience 106, 833-842.

Abramoff, M.D., Magalhaes, P.J., and Ram, S.J. (2004). Image Processing with ImageJ.

Biophotonics International 11, 36-42.

Afify, E.A. (2002). Turnover of mu-opioid receptors in neuroblastoma cells. Brain Res

Mol Brain Res 106, 83-87.

Akil, H., Owens, C., Gutstein, H., Taylor, L., Curran, E., and Watson, S. (1998).

Endogenous opioids: overview and current issues. Drug Alcohol Depend 51, 127-140.

Alfaras-Melainis, K., Gomes, I., Rozenfeld, R., Zachariou, V., and Devi, L. (2009).

Modulation of opioid receptor function by protein-protein interactions. Front Biosci 14,

3594-3607.

Ambrosio, E., Goldberg, S.R., and Elmer, G.I. (1995). Behavior genetic investigation of

the relationship between spontaneous locomotor activity and the acquisition of morphine

self-administration behavior. Behav Pharmacol 6, 229-237.

141

Ancevska-Taneva, N., Onoprishvili, I., Andria, M.L., Hiller, J.M., and Simon, E.J.

(2006). A member of the heat shock protein 40 family, hlj1, binds to the carboxyl tail of

the human mu opioid receptor. Brain Res 1081, 28-33.

Arttamangkul, S., Quillinan, N., Low, M.J., von Zastrow, M., Pintar, J., and Williams,

J.T. (2008). Differential activation and trafficking of micro-opioid receptors in brain

slices. Mol Pharmacol 74, 972-979.

Aubrun, F., Langeron, O., Quesnel, C., Coriat, P., and Riou, B. (2003). Relationships

between measurement of pain using visual analog score and morphine requirements

during postoperative intravenous morphine titration. Anesthesiology 98, 1415-1421.

Bailey, C.P., and Connor, M. (2005). Opioids: cellular mechanisms of tolerance and

physical dependence. Curr Opin Pharmacol 5, 60-68.

Ballesteros-Yanez, I., Ambrosio, E., Benavides-Piccione, R., Perez, J., Torres, I.,

Miguens, M., Garcia-Lecumberri, C., and DeFelipe, J. (2007). The effects of morphine

self-administration on cortical pyramidal cell structure in addiction-prone lewis rats.

Cereb Cortex 17, 238-249.

Ballesteros-Yanez, I., Ambrosio, E., Perez, J., Torres, I., Miguens, M., Garcia-

Lecumberri, C., and DeFelipe, J. (2008). Morphine self-administration effects on the

structure of cortical pyramidal cells in addiction-resistant rats. Brain Res 1230, 61-72.

Bare, L.A., Mansson, E., and Yang, D. (1994). Expression of two variants of the human

mu opioid receptor mRNA in SK-N-SH cells and human brain. FEBS Lett 354, 213-216.

Bart, G., Heilig, M., LaForge, K.S., Pollak, L., Leal, S.M., Ott, J., and Kreek, M.J.

(2004). Substantial attributable risk related to a functional mu-opioid receptor gene

polymorphism in association with heroin addiction in central Sweden. Mol Psychiatry 9,

547-549.

Bayerer, B., Stamer, U., Hoeft, A., and Stuber, F. (2007). Genomic variations and

transcriptional regulation of the human mu-opioid receptor gene. Eur J Pain 11, 421-427.

Becker, A., Grecksch, G., Brodemann, R., Kraus, J., Peters, B., Schroeder, H., Thiemann,

W., Loh, H.H., and Hollt, V. (2000). Morphine self-administration in mu-opioid receptor-

deficient mice. Naunyn Schmiedebergs Arch Pharmacol 361, 584-589.

Befort, K., Filliol, D., Decaillot, F.M., Gaveriaux-Ruff, C., Hoehe, M.R., and Kieffer,

B.L. (2001). A single nucleotide polymorphic mutation in the human mu-opioid receptor

severely impairs receptor signaling. J Biol Chem 276, 3130-3137.

Bennett, M.V. (1997). Gap junctions as electrical synapses. J Neurocytol 26, 349-366.

142

Berger, A.C., and Whistler, J.L. (2011). Morphine-induced mu opioid receptor trafficking

enhances reward yet prevents compulsive drug use. EMBO Mol Med 3, 385-397.

Berrendero, F., Kieffer, B.L., and Maldonado, R. (2002). Attenuation of nicotine-induced

antinociception, rewarding effects, and dependence in mu-opioid receptor knock-out

mice. J Neurosci 22, 10935-10940.

Berrettini, W.H., Alexander, R., Ferraro, T.N., and Vogel, W.H. (1994). A study of oral

morphine preference in inbred mouse strains. Psychiatr Genet 4, 81-86.

Bingol, B., and Sheng, M. (2011). Deconstruction for reconstruction: the role of

proteolysis in neural plasticity and disease. Neuron 69, 22-32.

Bloom, F.E. (1983). The endorphins: a growing family of pharmacologically pertinent

peptides. Annu Rev Pharmacol Toxicol 23, 151-170.

Blugeon, C., Le Crom, S., Richard, L., Vallat, J.M., Charnay, P., and Decker, L. (2011).

Dok4 is involved in Schwann cell myelination and axonal interaction in vitro. Glia 59,

351-362.

Bockaert, J., Perroy, J., Becamel, C., Marin, P., and Fagni, L. (2010). GPCR interacting

proteins (GIPs) in the nervous system: Roles in physiology and pathologies. Annu Rev

Pharmacol Toxicol 50, 89-109.

Bohn, L.M., Gainetdinov, R.R., Lin, F.T., Lefkowitz, R.J., and Caron, M.G. (2000). Mu-

opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not

dependence. Nature 408, 720-723.

Bolan, E.A., Pan, Y.X., and Pasternak, G.W. (2004). Functional analysis of MOR-1

splice variants of the mouse mu opioid receptor gene Oprm. Synapse 51, 11-18.

Bond, C., LaForge, K.S., Tian, M., Melia, D., Zhang, S., Borg, L., Gong, J., Schluger, J.,

Strong, J.A., Leal, S.M., et al. (1998). Single-nucleotide polymorphism in the human mu

opioid receptor gene alters beta-endorphin binding and activity: possible implications for

opiate addiction. Proc Natl Acad Sci U S A 95, 9608-9613.

Bousman, C.A., Chana, G., Glatt, S.J., Chandler, S.D., May, T., Lohr, J., Kremen, W.S.,

Tsuang, M.T., and Everall, I.P. (2010). Positive symptoms of psychosis correlate with

expression of ubiquitin proteasome genes in peripheral blood. Am J Med Genet B

Neuropsychiatr Genet 153B, 1336-1341.

Buday, L., and Tompa, P. (2010). Functional classification of scaffold proteins and

related molecules. FEBS J 277, 4348-4355.

143

Bursen, A., Moritz, S., Gaussmann, A., Dingermann, T., and Marschalek, R. (2004).

Interaction of AF4 wild-type and AF4.MLL fusion protein with SIAH proteins:

indication for t(4;11) pathobiology? Oncogene 23, 6237-6249.

Bushell, T., Endoh, T., Simen, A.A., Ren, D., Bindokas, V.P., and Miller, R.J. (2002).

Molecular components of tolerance to opiates in single hippocampal neurons. Mol

Pharmacol 61, 55-64.

Carthew, R.W., and Rubin, G.M. (1990). seven in absentia, a gene required for

specification of R7 cell fate in the Drosophila eye. Cell 63, 561-577.

Cen, B., Yu, Q., Guo, J., Wu, Y., Ling, K., Cheng, Z., Ma, L., and Pei, G. (2001). Direct

binding of beta-arrestins to two distinct intracellular domains of the delta opioid receptor.

J Neurochem 76, 1887-1894.

Chaipatikul, V., Erickson-Herbrandson, L.J., Loh, H.H., and Law, P.Y. (2003). Rescuing

the traffic-deficient mutants of rat mu-opioid receptors with hydrophobic ligands. Mol

Pharmacol 64, 32-41.

Chakrabarti, S., Liu, N.J., and Gintzler, A.R. (2010). Formation of mu-/kappa-opioid

receptor heterodimer is sex-dependent and mediates female-specific opioid analgesia.

Proc Natl Acad Sci U S A 107, 20115-20119.

Chang, A., Emmel, D.W., Rossi, G.C., and Pasternak, G.W. (1998). Methadone analgesia

in morphine-insensitive CXBK mice. Eur J Pharmacol 351, 189-191.

Charlton, J.J., Allen, P.B., Psifogeorgou, K., Chakravarty, S., Gomes, I., Neve, R.L.,

Devi, L.A., Greengard, P., Nestler, E.J., and Zachariou, V. (2008). Multiple actions of

spinophilin regulate mu opioid receptor function. Neuron 58, 238-247.

Chaturvedi, K., Bandari, P., Chinen, N., and Howells, R.D. (2001). Proteasome

involvement in agonist-induced down-regulation of mu and delta opioid receptors. J Biol

Chem 276, 12345-12355.

Chavkin, C., Bakhit, C., Weber, E., and Bloom, F.E. (1983). Relative contents and

concomitant release of prodynorphin/neoendorphin-derived peptides in rat hippocampus.

Proc Natl Acad Sci U S A 80, 7669-7673.

Chen, C., Li, J.G., Chen, Y., Huang, P., Wang, Y., and Liu-Chen, L.Y. (2006a). GEC1

interacts with the kappa opioid receptor and enhances expression of the receptor. J Biol

Chem 281, 7983-7993.

Chen, Y., Chen, C., Wang, Y., and Liu-Chen, L.Y. (2006b). Ligands regulate cell surface

level of the human kappa opioid receptor by activation-induced down-regulation and

pharmacological chaperone-mediated enhancement: differential effects of nonpeptide and

peptide agonists. J Pharmacol Exp Ther 319, 765-775.

144

Choo, Y.Y., Boh, B.K., Lou, J.J., Eng, J., Leck, Y.C., Anders, B., Smith, P.G., and

Hagen, T. (2011). Characterization of the role of COP9 signalosome in regulating cullin

E3 ubiquitin ligase activity. Mol Biol Cell 22, 4706-4715.

Chretien, M., Benjannet, S., Gossard, F., Gianoulakis, C., Crine, P., Lis, M., and Seidah,

N.G. (1979). From beta-lipotropin to beta-endorphin and 'pro-opio-melanocortin'. Can J

Biochem 57, 1111-1121.

Christie, M.J. (2008). Cellular neuroadaptations to chronic opioids: tolerance, withdrawal

and addiction. Br J Pharmacol 154, 384-396.

Collins, R.J., Weeks, J.R., Cooper, M.M., Good, P.I., and Russell, R.R. (1984).

Prediction of abuse liability of drugs using IV self-administration by rats.

Psychopharmacology (Berl) 82, 6-13.

Contarino, A., Picetti, R., Matthes, H.W., Koob, G.F., Kieffer, B.L., and Gold, L.H.

(2002). Lack of reward and locomotor stimulation induced by heroin in mu-opioid

receptor-deficient mice. Eur J Pharmacol 446, 103-109.

Cooke, S.F., and Bliss, T.V. (2006). Plasticity in the human central nervous system. Brain

129, 1659-1673.

Corbett, A.D., Henderson, G., McKnight, A.T., and Paterson, S.J. (2006). 75 years of

opioid research: the exciting but vain quest for the Holy Grail. Br J Pharmacol 147 Suppl

1, S153-162.

Crystal, H.A., Hamon, S., Randesi, M., Cook, J., Anastos, K., Lazar, J., Liu, C., Pearce,

L., Golub, E., Valcour, V., et al. (2012). A C17T polymorphism in the mu opiate receptor

is associated with quantitative measures of drug use in African American women. Addict

Biol 17, 181-191.

Cussac, D., Rauly-Lestienne, I., Heusler, P., Finana, F., Cathala, C., Bernois, S., and De

Vries, L. (2012). mu-opioid and 5-HT(1A) receptors heterodimerize and show signalling

crosstalk via G protein and MAP-kinase pathways. Cell Signal 24, 1648-1657.

Dacher, M., and Nugent, F.S. (2011). Opiates and plasticity. Neuropharmacology 61,

1088-1096.

Dahan, A., Sarton, E., Teppema, L., Olievier, C., Nieuwenhuijs, D., Matthes, H.W., and

Kieffer, B.L. (2001). Anesthetic potency and influence of morphine and sevoflurane on

respiration in mu-opioid receptor knockout mice. Anesthesiology 94, 824-832.

David, V., Matifas, A., Gavello-Baudy, S., Decorte, L., Kieffer, B.L., and Cazala, P.

(2008). Brain regional Fos expression elicited by the activation of mu- but not delta-

opioid receptors of the ventral tegmental area: evidence for an implication of the ventral

thalamus in opiate reward. Neuropsychopharmacology 33, 1746-1759.

145

De Cotiis, D.A., Woll, M.P., Fox, T.E., Hill, R.B., Levenson, R., and Flanagan, J.M.

(2008). Optimized expression and purification of myristoylated human neuronal calcium

sensor 1 in E. coli. Protein Expr Purif 61, 103-112.

Della, N.G., Senior, P.V., and Bowtell, D.D. (1993). Isolation and characterisation of

murine homologues of the Drosophila seven in absentia gene (sina). Development 117,

1333-1343.

Deng, W., Aimone, J.B., and Gage, F.H. (2010). New neurons and new memories: how

does adult hippocampal neurogenesis affect learning and memory? Nat Rev Neurosci 11,

339-350.

Depaux, A., Regnier-Ricard, F., Germani, A., and Varin-Blank, N. (2006). Dimerization

of hSiah proteins regulates their stability. Biochem Biophys Res Commun 348, 857-863.

Dhawan, B.N., Cesselin, F., Raghubir, R., Reisine, T., Bradley, P.B., Portoghese, P.S.,

and Hamon, M. (1996). International Union of Pharmacology. XII. Classification of

opioid receptors. Pharmacol Rev 48, 567-592.

Dickins, R.A., Frew, I.J., House, C.M., O'Bryan, M.K., Holloway, A.J., Haviv, I.,

Traficante, N., de Kretser, D.M., and Bowtell, D.D. (2002). The ubiquitin ligase

component Siah1a is required for completion of meiosis I in male mice. Mol Cell Biol

22, 2294-2303.

Dimitrova, Y.N., Li, J., Lee, Y.T., Rios-Esteves, J., Friedman, D.B., Choi, H.J., Weis,

W.I., Wang, C.Y., and Chazin, W.J. (2010). Direct ubiquitination of beta-catenin by

Siah-1 and regulation by the exchange factor TBL1. J Biol Chem 285, 13507-13516.

Doyle, G.A., Rebecca Sheng, X., Lin, S.S., Press, D.M., Grice, D.E., Buono, R.J.,

Ferraro, T.N., and Berrettini, W.H. (2007a). Identification of three mouse mu-opioid

receptor (MOR) gene (Oprm1) splice variants containing a newly identified alternatively

spliced exon. Gene 388, 135-147.

Doyle, G.A., Sheng, X.R., Lin, S.S., Press, D.M., Grice, D.E., Buono, R.J., Ferraro, T.N.,

and Berrettini, W.H. (2007b). Identification of five mouse mu-opioid receptor (MOR)

gene (Oprm1) splice variants containing a newly identified alternatively spliced exon.

Gene 395, 98-107.

Drake, M.T., Shenoy, S.K., and Lefkowitz, R.J. (2006). Trafficking of G protein-coupled

receptors. Circ Res 99, 570-582.

Drakenberg, K., Nikoshkov, A., Horvath, M.C., Fagergren, P., Gharibyan, A.,

Saarelainen, K., Rahman, S., Nylander, I., Bakalkin, G., Rajs, J., et al. (2006). Mu opioid

receptor A118G polymorphism in association with striatal opioid neuropeptide gene

expression in heroin abusers. Proc Natl Acad Sci U S A 103, 7883-7888.

146

Eisch, A.J., Barrot, M., Schad, C.A., Self, D.W., and Nestler, E.J. (2000). Opiates inhibit

neurogenesis in the adult rat hippocampus. Proc Natl Acad Sci U S A 97, 7579-7584.

Elmer, G.I., Pieper, J.O., Hamilton, L.R., and Wise, R.A. (2010). Qualitative differences

between C57BL/6J and DBA/2J mice in morphine potentiation of brain stimulation

reward and intravenous self-administration. Psychopharmacology (Berl) 208, 309-321.

Famulski, J.K., Trivedi, N., Howell, D., Yang, Y., Tong, Y., Gilbertson, R., and Solecki,

D.J. (2010). Siah regulation of Pard3A controls neuronal cell adhesion during germinal

zone exit. Science 330, 1834-1838.

Feldman, M., and van der Goot, F.G. (2009). Novel ubiquitin-dependent quality control

in the endoplasmic reticulum. Trends Cell Biol 19, 357-363.

Feng, G.J., Kellett, E., Scorer, C.A., Wilde, J., White, J.H., and Milligan, G. (2003).

Selective interactions between helix VIII of the human mu-opioid receptors and the C

terminus of periplakin disrupt G protein activation. J Biol Chem 278, 33400-33407.

Fields, S., and Song, O. (1989). A novel genetic system to detect protein-protein

interactions. Nature 340, 245-246.

Fillingim, R.B., Kaplan, L., Staud, R., Ness, T.J., Glover, T.L., Campbell, C.M., Mogil,

J.S., and Wallace, M.R. (2005). The A118G single nucleotide polymorphism of the mu-

opioid receptor gene (OPRM1) is associated with pressure pain sensitivity in humans. J

Pain 6, 159-167.

Fillingim, R.B., Wallace, M.R., Herbstman, D.M., Ribeiro-Dasilva, M., and Staud, R.

(2008). Genetic contributions to pain: a review of findings in humans. Oral Dis 14, 673-

682.

Filliol, D., Ghozland, S., Chluba, J., Martin, M., Matthes, H.W., Simonin, F., Befort, K.,

Gaveriaux-Ruff, C., Dierich, A., LeMeur, M., et al. (2000). Mice deficient for delta- and

mu-opioid receptors exhibit opposing alterations of emotional responses. Nat Genet 25,

195-200.

Finn, A.K., and Whistler, J.L. (2001). Endocytosis of the mu opioid receptor reduces

tolerance and a cellular hallmark of opiate withdrawal. Neuron 32, 829-839.

Frew, I.J., Hammond, V.E., Dickins, R.A., Quinn, J.M., Walkley, C.R., Sims, N.A.,

Schnall, R., Della, N.G., Holloway, A.J., Digby, M.R., et al. (2003). Generation and

analysis of Siah2 mutant mice. Mol Cell Biol 23, 9150-9161.

Gainetdinov, R.R., Premont, R.T., Bohn, L.M., Lefkowitz, R.J., and Caron, M.G. (2004).

Desensitization of G protein-coupled receptors and neuronal functions. Annu Rev

Neurosci 27, 107-144.

147

Garzon, J., Rodriguez-Munoz, M., de la Torre-Madrid, E., and Sanchez-Blazquez, P.

(2005a). Effector antagonism by the regulators of G protein signalling (RGS) proteins

causes desensitization of mu-opioid receptors in the CNS. Psychopharmacology (Berl)

180, 1-11.

Garzon, J., Rodriguez-Munoz, M., and Sanchez-Blazquez, P. (2005b). Morphine alters

the selective association between mu-opioid receptors and specific RGS proteins in

mouse periaqueductal gray matter. Neuropharmacology 48, 853-868.

Gaveriaux-Ruff, C., and Kieffer, B.L. (2002). Opioid receptor genes inactivated in mice:

the highlights. Neuropeptides 36, 62-71.

Ge, X., Qiu, Y., Loh, H.H., and Law, P.Y. (2009). GRIN1 regulates micro-opioid

receptor activities by tethering the receptor and G protein in the lipid raft. J Biol Chem

284, 36521-36534.

Gelernter, J., Kranzler, H., and Cubells, J. (1999). Genetics of two mu opioid receptor

gene (OPRM1) exon I polymorphisms: population studies, and allele frequencies in

alcohol- and drug-dependent subjects. Mol Psychiatry 4, 476-483.

George, S.R., Fan, T., Xie, Z., Tse, R., Tam, V., Varghese, G., and O'Dowd, B.F. (2000).

Oligomerization of mu- and delta-opioid receptors. Generation of novel functional

properties. J Biol Chem 275, 26128-26135.

George, S.R., O'Dowd, B.F., and Lee, S.P. (2002). G-protein-coupled receptor

oligomerization and its potential for drug discovery. Nat Rev Drug Discov 1, 808-820.

Georgoussi, Z., Georganta, E.M., and Milligan, G. (2012). The other side of opioid

receptor signalling: regulation by protein-protein interaction. Curr Drug Targets 13, 80-

102.

Georgoussi, Z., Leontiadis, L., Mazarakou, G., Merkouris, M., Hyde, K., and Hamm, H.

(2006). Selective interactions between G protein subunits and RGS4 with the C-terminal

domains of the mu- and delta-opioid receptors regulate opioid receptor signaling. Cell

Signal 18, 771-782.

Georgoussi, Z., Merkouris, M., Mullaney, I., Megaritis, G., Carr, C., Zioudrou, C., and

Milligan, G. (1997). Selective interactions of mu-opioid receptors with pertussis toxin-

sensitive G proteins: involvement of the third intracellular loop and the c-terminal tail in

coupling. Biochim Biophys Acta 1359, 263-274.

Germani, A., Bruzzoni-Giovanelli, H., Fellous, A., Gisselbrecht, S., Varin-Blank, N., and

Calvo, F. (2000). SIAH-1 interacts with alpha-tubulin and degrades the kinesin Kid by

the proteasome pathway during mitosis. Oncogene 19, 5997-6006.

148

Germani, A., Romero, F., Houlard, M., Camonis, J., Gisselbrecht, S., Fischer, S., and

Varin-Blank, N. (1999). hSiah2 is a new Vav binding protein which inhibits Vav-

mediated signaling pathways. Mol Cell Biol 19, 3798-3807.

Ghitza, U.E., Zhai, H., Wu, P., Airavaara, M., Shaham, Y., and Lu, L. (2010). Role of

BDNF and GDNF in drug reward and relapse: a review. Neurosci Biobehav Rev 35, 157-

171.

Ghozland, S., Matthes, H.W., Simonin, F., Filliol, D., Kieffer, B.L., and Maldonado, R.

(2002). Motivational effects of cannabinoids are mediated by mu-opioid and kappa-

opioid receptors. J Neurosci 22, 1146-1154.

Ginosar, Y., Davidson, E.M., Meroz, Y., Blotnick, S., Shacham, M., and Caraco, Y.

(2009). Mu-opioid receptor (A118G) single-nucleotide polymorphism affects alfentanil

requirements for extracorporeal shock wave lithotripsy: a pharmacokinetic-

pharmacodynamic study. Br J Anaesth 103, 420-427.

Glatt, S.J., Bousman, C., Wang, R.S., Murthy, K.K., Rana, B.K., Lasky-Su, J.A., Zhu,

S.C., Zhang, R., Li, J., Zhang, B., et al. (2007). Evaluation of OPRM1 variants in heroin

dependence by family-based association testing and meta-analysis. Drug Alcohol Depend

90, 159-165.

Gomes, I., Gupta, A., Filipovska, J., Szeto, H.H., Pintar, J.E., and Devi, L.A. (2004). A

role for heterodimerization of mu and delta opiate receptors in enhancing morphine

analgesia. Proc Natl Acad Sci U S A 101, 5135-5139.

Gomes, I., Jordan, B.A., Gupta, A., Trapaidze, N., Nagy, V., and Devi, L.A. (2000).

Heterodimerization of mu and delta opioid receptors: A role in opiate synergy. J Neurosci

20, RC110.

Griffiths, R.R. (1980). Common factors in human and infrahuman drug self-

administration. Psychopharmacol Bull 16, 45-47.

Groer, C.E., Schmid, C.L., Jaeger, A.M., and Bohn, L.M. (2011). Agonist-directed

interactions with specific beta-arrestins determine mu-opioid receptor trafficking,

ubiquitination, and dephosphorylation. J Biol Chem 286, 31731-31741.

Guang, W., Wang, H., Su, T., Weinstein, I.B., and Wang, J.B. (2004). Role of mPKCI, a

novel mu-opioid receptor interactive protein, in receptor desensitization, phosphorylation,

and morphine-induced analgesia. Mol Pharmacol 66, 1285-1292.

Haberstock-Debic, H., Kim, K.A., Yu, Y.J., and von Zastrow, M. (2005). Morphine

promotes rapid, arrestin-dependent endocytosis of mu-opioid receptors in striatal

neurons. J Neurosci 25, 7847-7857.

149

Haberstock-Debic, H., Wein, M., Barrot, M., Colago, E.E., Rahman, Z., Neve, R.L.,

Pickel, V.M., Nestler, E.J., von Zastrow, M., and Svingos, A.L. (2003). Morphine acutely

regulates opioid receptor trafficking selectively in dendrites of nucleus accumbens

neurons. J Neurosci 23, 4324-4332.

Haefliger, J.A., Bruzzone, R., Jenkins, N.A., Gilbert, D.J., Copeland, N.G., and Paul,

D.L. (1992). Four novel members of the connexin family of gap junction proteins.

Molecular cloning, expression, and chromosome mapping. J Biol Chem 267, 2057-2064.

Haklai-Topper, L., Mlechkovich, G., Savariego, D., Gokhman, I., and Yaron, A. (2010).

Cis interaction between Semaphorin6A and Plexin-A4 modulates the repulsive response

to Sema6A. EMBO J 29, 2635-2645.

Hamm, H.E., and Gilchrist, A. (1996). Heterotrimeric G proteins. Curr Opin Cell Biol 8,

189-196.

Harburg, G.C., Hall, F.S., Harrist, A.V., Sora, I., Uhl, G.R., and Eisch, A.J. (2007).

Knockout of the mu opioid receptor enhances the survival of adult-generated

hippocampal granule cell neurons. Neuroscience 144, 77-87.

Harrison, L.M., Kastin, A.J., and Zadina, J.E. (1998). Opiate tolerance and dependence:

receptors, G-proteins, and antiopiates. Peptides 19, 1603-1630.

Hausmann, G., Banziger, C., and Basler, K. (2007). Helping Wingless take flight: how

WNT proteins are secreted. Nat Rev Mol Cell Biol 8, 331-336.

He, L., Fong, J., von Zastrow, M., and Whistler, J.L. (2002). Regulation of opioid

receptor trafficking and morphine tolerance by receptor oligomerization. Cell 108, 271-

282.

He, L., Kim, J.A., and Whistler, J.L. (2009). Biomarkers of morphine tolerance and

dependence are prevented by morphine-induced endocytosis of a mutant mu-opioid

receptor. FASEB J 23, 4327-4334.

Henry, A.G., White, I.J., Marsh, M., von Zastrow, M., and Hislop, J.N. (2011). The role

of ubiquitination in lysosomal trafficking of delta-opioid receptors. Traffic 12, 170-184.

Hicke, L., and Dunn, R. (2003). Regulation of membrane protein transport by ubiquitin

and ubiquitin-binding proteins. Annu Rev Cell Dev Biol 19, 141-172.

Hill, S.J. (2006). G-protein-coupled receptors: past, present and future. Br J Pharmacol

147 Suppl 1, S27-37.

Hislop, J.N., Henry, A.G., Marchese, A., and von Zastrow, M. (2009). Ubiquitination

regulates proteolytic processing of G protein-coupled receptors after their sorting to

lysosomes. J Biol Chem 284, 19361-19370.

150

Hislop, J.N., Henry, A.G., and von Zastrow, M. (2011). Ubiquitination in the first

cytoplasmic loop of mu-opioid receptors reveals a hierarchical mechanism of lysosomal

down-regulation. J Biol Chem 286, 40193-40204.

Hislop, J.N., and von Zastrow, M. (2011). Role of ubiquitination in endocytic trafficking

of G-protein-coupled receptors. Traffic 12, 137-148.

Hoehe, M.R., Kopke, K., Wendel, B., Rohde, K., Flachmeier, C., Kidd, K.K., Berrettini,

W.H., and Church, G.M. (2000). Sequence variability and candidate gene analysis in

complex disease: association of mu opioid receptor gene variation with substance

dependence. Hum Mol Genet 9, 2895-2908.

House, C.M., Frew, I.J., Huang, H.L., Wiche, G., Traficante, N., Nice, E., Catimel, B.,

and Bowtell, D.D. (2003). A binding motif for Siah ubiquitin ligase. Proc Natl Acad Sci

U S A 100, 3101-3106.

Hu, G., Chung, Y.L., Glover, T., Valentine, V., Look, A.T., and Fearon, E.R. (1997a).

Characterization of human homologs of the Drosophila seven in absentia (sina) gene.

Genomics 46, 103-111.

Hu, G., and Fearon, E.R. (1999). Siah-1 N-terminal RING domain is required for

proteolysis function, and C-terminal sequences regulate oligomerization and binding to

target proteins. Mol Cell Biol 19, 724-732.

Hu, G., Zhang, S., Vidal, M., Baer, J.L., Xu, T., and Fearon, E.R. (1997b). Mammalian

homologs of seven in absentia regulate DCC via the ubiquitin-proteasome pathway.

Genes Dev 11, 2701-2714.

Huang, C.J., Liu, H.F., Su, N.Y., Hsu, Y.W., Yang, C.H., Chen, C.C., and Tsai, P.S.

(2008). Association between human opioid receptor genes polymorphisms and pressure

pain sensitivity in females*. Anaesthesia 63, 1288-1295.

Huang, P., and Liu-Chen, L.Y. (2009). Detecting the mu opioid receptor in brain

following SDS-PAGE with multiple approaches. Front Biosci (Elite Ed) 1, 220-227.

Hyman, S.E. (2007). The neurobiology of addiction: implications for voluntary control of

behavior. Am J Bioeth 7, 8-11.

Ide, S., Han, W., Kasai, S., Hata, H., Sora, I., and Ikeda, K. (2005). Characterization of

the 3' untranslated region of the human mu-opioid receptor (MOR-1) mRNA. Gene 364,

139-145.

Ikeda, K., Ichikawa, T., Kobayashi, T., Kumanishi, T., Oike, S., and Yano, R. (1999).

Unique behavioural phenotypes of recombinant-inbred CXBK mice: partial deficiency of

sensitivity to mu- and kappa-agonists. Neurosci Res 34, 149-155.

151

Ikeda, K., Kobayashi, T., Ichikawa, T., Kumanishi, T., Niki, H., and Yano, R. (2001).

The untranslated region of (mu)-opioid receptor mRNA contributes to reduced opioid

sensitivity in CXBK mice. J Neurosci 21, 1334-1339.

Imai, Y., Soda, M., Hatakeyama, S., Akagi, T., Hashikawa, T., Nakayama, K.I., and

Takahashi, R. (2002). CHIP is associated with Parkin, a gene responsible for familial

Parkinson's disease, and enhances its ubiquitin ligase activity. Mol Cell 10, 55-67.

Inturrisi, C.E. (2002). Clinical pharmacology of opioids for pain. Clin J Pain 18, S3-13.

Ishikawa, K., Nash, S.R., Nishimune, A., Neki, A., Kaneko, S., and Nakanishi, S. (1999).

Competitive interaction of seven in absentia homolog-1A and Ca2+/calmodulin with the

cytoplasmic tail of group 1 metabotropic glutamate receptors. Genes Cells 4, 381-390.

Iyer, K., Burkle, L., Auerbach, D., Thaminy, S., Dinkel, M., Engels, K., and Stagljar, I.

(2005). Utilizing the split-ubiquitin membrane yeast two-hybrid system to identify

protein-protein interactions of integral membrane proteins. Sci STKE 2005, pl3.

Jin, J., Kittanakom, S., Wong, V., Reyes, B.A., Van Bockstaele, E.J., Stagljar, I.,

Berrettini, W., and Levenson, R. (2010). Interaction of the mu-opioid receptor with

GPR177 (Wntless) inhibits Wnt secretion: potential implications for opioid dependence.

BMC Neurosci 11, 33.

Johnson, E.E., Christie, M.J., and Connor, M. (2005). The role of opioid receptor

phosphorylation and trafficking in adaptations to persistent opioid treatment.

Neurosignals 14, 290-302.

Johnson, P.S., Wang, J.B., Wang, W.F., and Uhl, G.R. (1994). Expressed mu opiate

receptor couples to adenylate cyclase and phosphatidyl inositol turnover. Neuroreport 5,

507-509.

Jordan, B.A., and Devi, L.A. (1999). G-protein-coupled receptor heterodimerization

modulates receptor function. Nature 399, 697-700.

Jordan, B.A., Gomes, I., Rios, C., Filipovska, J., and Devi, L.A. (2003). Functional

interactions between mu opioid and alpha 2A-adrenergic receptors. Mol Pharmacol 64,

1317-1324.

Kabbani, N., and Levenson, R. (2007). A proteomic approach to receptor signaling:

molecular mechanisms and therapeutic implications derived from discovery of the

dopamine D2 receptor signalplex. Eur J Pharmacol 572, 83-93.

Kabbani, N., Negyessy, L., Lin, R., Goldman-Rakic, P., and Levenson, R. (2002).

Interaction with neuronal calcium sensor NCS-1 mediates desensitization of the D2

dopamine receptor. J Neurosci 22, 8476-8486.

152

Kapur, S., Sharad, S., Singh, R.A., and Gupta, A.K. (2007). A118g polymorphism in mu

opioid receptor gene (oprm1): association with opiate addiction in subjects of Indian

origin. J Integr Neurosci 6, 511-522.

Kasai, S., and Ikeda, K. (2011). Pharmacogenomics of the human micro-opioid receptor.

Pharmacogenomics 12, 1305-1320.

Kato, A., Kawamata, N., Tamayose, K., Egashira, M., Miura, R., Fujimura, T.,

Murayama, K., and Oshimi, K. (2002). Ancient ubiquitous protein 1 binds to the

conserved membrane-proximal sequence of the cytoplasmic tail of the integrin alpha

subunits that plays a crucial role in the inside-out signaling of alpha IIbbeta 3. J Biol

Chem 277, 28934-28941.

Keith, D.E., Murray, S.R., Zaki, P.A., Chu, P.C., Lissin, D.V., Kang, L., Evans, C.J., and

von Zastrow, M. (1996). Morphine activates opioid receptors without causing their rapid

internalization. J Biol Chem 271, 19021-19024.

Kieffer, B.L., and Gaveriaux-Ruff, C. (2002). Exploring the opioid system by gene

knockout. Prog Neurobiol 66, 285-306.

Kitanaka, N., Sora, I., Kinsey, S., Zeng, Z., and Uhl, G.R. (1998). No heroin or morphine

6beta-glucuronide analgesia in mu-opioid receptor knockout mice. Eur J Pharmacol 355,

R1-3.

Kittanakom, S., Chuk, M., Wong, V., Snyder, J., Edmonds, D., Lydakis, A., Zhang, Z.,

Auerbach, D., and Stagljar, I. (2009). Analysis of membrane protein complexes using the

split-ubiquitin membrane yeast two-hybrid (MYTH) system. Methods Mol Biol 548, 247-

271.

Knapp, R.J., Malatynska, E., Fang, L., Li, X., Babin, E., Nguyen, M., Santoro, G., Varga,

E.V., Hruby, V.J., Roeske, W.R., et al. (1994). Identification of a human delta opioid

receptor: cloning and expression. Life Sci 54, PL463-469.

Koch, T., Brandenburg, L.O., Schulz, S., Liang, Y., Klein, J., and Hollt, V. (2003). ADP-

ribosylation factor-dependent phospholipase D2 activation is required for agonist-induced

mu-opioid receptor endocytosis. J Biol Chem 278, 9979-9985.

Koch, T., Kroslak, T., Averbeck, M., Mayer, P., Schroder, H., Raulf, E., and Hollt, V.

(2000). Allelic variation S268P of the human mu-opioid receptor affects both

desensitization and G protein coupling. Mol Pharmacol 58, 328-334.

Kong, H., Raynor, K., Yano, H., Takeda, J., Bell, G.I., and Reisine, T. (1994). Agonists

and antagonists bind to different domains of the cloned kappa opioid receptor. Proc Natl

Acad Sci U S A 91, 8042-8046.

153

Kostova, Z., Tsai, Y.C., and Weissman, A.M. (2007). Ubiquitin ligases, critical mediators

of endoplasmic reticulum-associated degradation. Semin Cell Dev Biol 18, 770-779.

Kramer, J.W., Post, M.A., Brown, A.M., and Kirsch, G.E. (1998). Modulation of

potassium channel gating by coexpression of Kv2.1 with regulatory Kv5.1 or Kv6.1

alpha-subunits. Am J Physiol 274, C1501-1510.

Kreek, M.J., Bart, G., Lilly, C., LaForge, K.S., and Nielsen, D.A. (2005).

Pharmacogenetics and human molecular genetics of opiate and cocaine addictions and

their treatments. Pharmacol Rev 57, 1-26.

Kroslak, T., Laforge, K.S., Gianotti, R.J., Ho, A., Nielsen, D.A., and Kreek, M.J. (2007).

The single nucleotide polymorphism A118G alters functional properties of the human mu

opioid receptor. J Neurochem 103, 77-87.

Lachman, H.M. (2006). An overview of the genetics of substance use disorders. Curr

Psychiatry Rep 8, 133-143.

Lamprecht, R., and LeDoux, J. (2004). Structural plasticity and memory. Nat Rev

Neurosci 5, 45-54.

Lasagna, L. (1954). Nalorphine (n-allylnormorphine); practical and theoretical

considerations. AMA Arch Intern Med 94, 532-558.

Law, P.Y., Erickson, L.J., El-Kouhen, R., Dicker, L., Solberg, J., Wang, W., Miller, E.,

Burd, A.L., and Loh, H.H. (2000). Receptor density and recycling affect the rate of

agonist-induced desensitization of mu-opioid receptor. Mol Pharmacol 58, 388-398.

Le Merrer, J., Becker, J.A., Befort, K., and Kieffer, B.L. (2009). Reward processing by

the opioid system in the brain. Physiol Rev 89, 1379-1412.

Lentze, N., and Auerbach, D. (2008). Membrane-based yeast two-hybrid system to detect

protein interactions. Curr Protoc Protein Sci Chapter 19, Unit 19 17.

Li, J.G., Haines, D.S., and Liu-Chen, L.Y. (2008). Agonist-promoted Lys63-linked

polyubiquitination of the human kappa-opioid receptor is involved in receptor down-

regulation. Mol Pharmacol 73, 1319-1330.

Li, Y., Chen, J., Bai, B., Du, H., Liu, Y., and Liu, H. (2012). Heterodimerization of

human apelin and kappa opioid receptors: roles in signal transduction. Cell Signal 24,

991-1001.

Liang, Y.J., Wu, D.F., Yang, L.Q., Hollt, V., and Koch, T. (2007). Interaction of the mu-

opioid receptor with synaptophysin influences receptor trafficking and signaling. Mol

Pharmacol 71, 123-131.

154

Liao, D., Lin, H., Law, P.Y., and Loh, H.H. (2005). Mu-opioid receptors modulate the

stability of dendritic spines. Proc Natl Acad Sci U S A 102, 1725-1730.

Lie, D.C., Colamarino, S.A., Song, H.J., Desire, L., Mira, H., Consiglio, A., Lein, E.S.,

Jessberger, S., Lansford, H., Dearie, A.R., et al. (2005). Wnt signalling regulates adult

hippocampal neurogenesis. Nature 437, 1370-1375.

Lin, R., Karpa, K., Kabbani, N., Goldman-Rakic, P., and Levenson, R. (2001). Dopamine

D2 and D3 receptors are linked to the actin cytoskeleton via interaction with filamin A.

Proc Natl Acad Sci U S A 98, 5258-5263.

Lin R, K.N., Binda A, Canfield V, Levenson R (2005). Use of the yeast two-hybrid

system to detect receptor-protein interactions. In G-Protein-Coupled Receptor--Protein

Interactions, G.S.a.O.D. BF, ed. (John Wiley & Sons, Inc.).

Liu, J., Stevens, J., Rote, C.A., Yost, H.J., Hu, Y., Neufeld, K.L., White, R.L., and

Matsunami, N. (2001). Siah-1 mediates a novel beta-catenin degradation pathway linking

p53 to the adenomatous polyposis coli protein. Mol Cell 7, 927-936.

Liu, Y., Shah, S.V., Xiang, X., Wang, J., Deng, Z.B., Liu, C., Zhang, L., Wu, J.,

Edmonds, T., Jambor, C., et al. (2009). COP9-associated CSN5 regulates exosomal

protein deubiquitination and sorting. Am J Pathol 174, 1415-1425.

Lotsch, J., Zimmermann, M., Darimont, J., Marx, C., Dudziak, R., Skarke, C., and

Geisslinger, G. (2002). Does the A118G polymorphism at the mu-opioid receptor gene

protect against morphine-6-glucuronide toxicity? Anesthesiology 97, 814-819.

Luo, W., Wang, Y., Hanck, T., Stricker, R., and Reiser, G. (2006). Jab1, a novel

protease-activated receptor-2 (PAR-2)-interacting protein, is involved in PAR-2-induced

activation of activator protein-1. J Biol Chem 281, 7927-7936.

Lynch, W.J., Nicholson, K.L., Dance, M.E., Morgan, R.W., and Foley, P.L. (2010).

Animal models of substance abuse and addiction: implications for science, animal

welfare, and society. Comp Med 60, 177-188.

Mague, S.D., and Blendy, J.A. (2010). OPRM1 SNP (A118G): involvement in disease

development, treatment response, and animal models. Drug Alcohol Depend 108, 172-

182.

Majumdar, S., Grinnell, S., Le Rouzic, V., Burgman, M., Polikar, L., Ansonoff, M.,

Pintar, J., Pan, Y.X., and Pasternak, G.W. (2011). Truncated G protein-coupled mu

opioid receptor MOR-1 splice variants are targets for highly potent opioid analgesics

lacking side effects. Proc Natl Acad Sci U S A 108, 19778-19783.

Marchese, A., Paing, M.M., Temple, B.R., and Trejo, J. (2008). G protein-coupled

receptor sorting to endosomes and lysosomes. Annu Rev Pharmacol Toxicol 48, 601-629.

155

Marcol, W., Kotulska, K., Larysz-Brysz, M., and Kowalik, J.L. (2007). BDNF

contributes to animal model neuropathic pain after peripheral nerve transection.

Neurosurg Rev 30, 235-243; discussion 243.

Martin, W.R., Eades, C.G., Thompson, J.A., Huppler, R.E., and Gilbert, P.E. (1976). The

effects of morphine- and nalorphine- like drugs in the nondependent and morphine-

dependent chronic spinal dog. J Pharmacol Exp Ther 197, 517-532.

Martini, L., and Whistler, J.L. (2007). The role of mu opioid receptor desensitization and

endocytosis in morphine tolerance and dependence. Curr Opin Neurobiol 17, 556-564.

Mathon, D.S., Lesscher, H.M., Gerrits, M.A., Kamal, A., Pintar, J.E., Schuller, A.G.,

Spruijt, B.M., Burbach, J.P., Smidt, M.P., van Ree, J.M., et al. (2005). Increased

gabaergic input to ventral tegmental area dopaminergic neurons associated with

decreased cocaine reinforcement in mu-opioid receptor knockout mice. Neuroscience

130, 359-367.

Matsuzawa, S., Takayama, S., Froesch, B.A., Zapata, J.M., and Reed, J.C. (1998). p53-

inducible human homologue of Drosophila seven in absentia (Siah) inhibits cell growth:

suppression by BAG-1. EMBO J 17, 2736-2747.

Matthes, H.W., Maldonado, R., Simonin, F., Valverde, O., Slowe, S., Kitchen, I., Befort,

K., Dierich, A., Le Meur, M., Dolle, P., et al. (1996). Loss of morphine-induced

analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioid-

receptor gene. Nature 383, 819-823.

Matthes, H.W., Smadja, C., Valverde, O., Vonesch, J.L., Foutz, A.S., Boudinot, E.,

Denavit-Saubie, M., Severini, C., Negri, L., Roques, B.P., et al. (1998). Activity of the

delta-opioid receptor is partially reduced, whereas activity of the kappa-receptor is

maintained in mice lacking the mu-receptor. J Neurosci 18, 7285-7295.

Mazarakou, G., and Georgoussi, Z. (2005). STAT5A interacts with and is phosphorylated

upon activation of the mu-opioid receptor. J Neurochem 93, 918-931.

Mehnert, M., Sommer, T., and Jarosch, E. (2010). ERAD ubiquitin ligases:

multifunctional tools for protein quality control and waste disposal in the endoplasmic

reticulum. Bioessays 32, 905-913.

Miller, J., and Stagljar, I. (2004). Using the yeast two-hybrid system to identify

interacting proteins. Methods Mol Biol 261, 247-262.

Milligan, G. (2005). Opioid receptors and their interacting proteins. Neuromolecular Med

7, 51-59.

Minami, M., and Satoh, M. (1995). Molecular biology of the opioid receptors: structures,

functions and distributions. Neurosci Res 23, 121-145.

156

Moles, A., Kieffer, B.L., and D'Amato, F.R. (2004). Deficit in attachment behavior in

mice lacking the mu-opioid receptor gene. Science 304, 1983-1986.

Molinari, P., Vezzi, V., Sbraccia, M., Gro, C., Riitano, D., Ambrosio, C., Casella, I., and

Costa, T. (2010). Morphine-like opiates selectively antagonize receptor-arrestin

interactions. J Biol Chem 285, 12522-12535.

Mollereau, C., Simons, M.J., Soularue, P., Liners, F., Vassart, G., Meunier, J.C., and

Parmentier, M. (1996). Structure, tissue distribution, and chromosomal localization of the

prepronociceptin gene. Proc Natl Acad Sci U S A 93, 8666-8670.

Moriyoshi, K., Iijima, K., Fujii, H., Ito, H., Cho, Y., and Nakanishi, S. (2004). Seven in

absentia homolog 1A mediates ubiquitination and degradation of group 1 metabotropic

glutamate receptors. Proc Natl Acad Sci U S A 101, 8614-8619.

Moser, P., Wolinsky, T., Castagne, V., and Duxon, M. (2011). Current approaches and

issues in non-clinical evaluation of abuse and dependence. J Pharmacol Toxicol Methods

63, 160-167.

Mueller, B., Klemm, E.J., Spooner, E., Claessen, J.H., and Ploegh, H.L. (2008). SEL1L

nucleates a protein complex required for dislocation of misfolded glycoproteins. Proc

Natl Acad Sci U S A 105, 12325-12330.

Myers, K.M., Bechtholt-Gompf, A.J., Coleman, B.R., and Carlezon, W.A., Jr. (2012).

Extinction of conditioned opiate withdrawal in rats in a two-chambered place

conditioning apparatus. Nat Protoc 7, 517-526.

Myung, J., Kim, K.B., and Crews, C.M. (2001). The ubiquitin-proteasome pathway and

proteasome inhibitors. Med Res Rev 21, 245-273.

Neil, A. (1984). Affinities of some common opioid analgesics towards four binding sites

in mouse brain. Naunyn Schmiedebergs Arch Pharmacol 328, 24-29.

Nemani, M., Linares-Cruz, G., Bruzzoni-Giovanelli, H., Roperch, J.P., Tuynder, M.,

Bougueleret, L., Cherif, D., Medhioub, M., Pasturaud, P., Alvaro, V., et al. (1996).

Activation of the human homologue of the Drosophila sina gene in apoptosis and tumor

suppression. Proc Natl Acad Sci U S A 93, 9039-9042.

Nestler, E.J. (2001). Molecular neurobiology of addiction. Am J Addict 10, 201-217.

Nestler, E.J. (2004). Molecular mechanisms of drug addiction. Neuropharmacology 47

Suppl 1, 24-32.

Nugent, F.S., Penick, E.C., and Kauer, J.A. (2007). Opioids block long-term potentiation

of inhibitory synapses. Nature 446, 1086-1090.

157

Obata, K., and Noguchi, K. (2006). BDNF in sensory neurons and chronic pain. Neurosci

Res 55, 1-10.

Oldfield, S., Braksator, E., Rodriguez-Martin, I., Bailey, C.P., Donaldson, L.F.,

Henderson, G., and Kelly, E. (2008). C-terminal splice variants of the mu-opioid

receptor: existence, distribution and functional characteristics. J Neurochem 104, 937-

945.

Oliver, P.L., Bitoun, E., Clark, J., Jones, E.L., and Davies, K.E. (2004). Mediation of Af4

protein function in the cerebellum by Siah proteins. Proc Natl Acad Sci U S A 101,

14901-14906.

Onogi, T., Minami, M., Katao, Y., Nakagawa, T., Aoki, Y., Toya, T., Katsumata, S., and

Satoh, M. (1995). DAMGO, a mu-opioid receptor selective agonist, distinguishes

between mu- and delta-opioid receptors around their first extracellular loops. FEBS Lett

357, 93-97.

Onoprishvili, I., Andria, M.L., Kramer, H.K., Ancevska-Taneva, N., Hiller, J.M., and

Simon, E.J. (2003). Interaction between the mu opioid receptor and filamin A is involved

in receptor regulation and trafficking. Mol Pharmacol 64, 1092-1100.

Pan, L., Xu, J., Yu, R., Xu, M.M., Pan, Y.X., and Pasternak, G.W. (2005). Identification

and characterization of six new alternatively spliced variants of the human mu opioid

receptor gene, Oprm. Neuroscience 133, 209-220.

Pan, Y.X. (2005). Diversity and complexity of the mu opioid receptor gene: alternative

pre-mRNA splicing and promoters. DNA Cell Biol 24, 736-750.

Pan, Y.X., Xu, J., Mahurter, L., Bolan, E., Xu, M., and Pasternak, G.W. (2001).

Generation of the mu opioid receptor (MOR-1) protein by three new splice variants of the

Oprm gene. Proc Natl Acad Sci U S A 98, 14084-14089.

Pan, Y.X., Xu, J., Mahurter, L., Xu, M., Gilbert, A.K., and Pasternak, G.W. (2003).

Identification and characterization of two new human mu opioid receptor splice variants,

hMOR-1O and hMOR-1X. Biochem Biophys Res Commun 301, 1057-1061.

Pardon, M.C. (2010). Role of neurotrophic factors in behavioral processes: implications

for the treatment of psychiatric and neurodegenerative disorders. Vitam Horm 82, 185-

200.

Parenty, G., Appelbe, S., and Milligan, G. (2008). CXCR2 chemokine receptor

antagonism enhances DOP opioid receptor function via allosteric regulation of the

CXCR2-DOP receptor heterodimer. Biochem J 412, 245-256.

Pasquier, C., Promponas, V.J., Palaios, G.A., Hamodrakas, J.S., and Hamodrakas, S.J.

(1999). A novel method for predicting transmembrane segments in proteins based on a

158

statistical analysis of the SwissProt database: the PRED-TMR algorithm. Protein Eng 12,

381-385.

Pasternak, D.A., Pan, L., Xu, J., Yu, R., Xu, M.M., Pasternak, G.W., and Pan, Y.X.

(2004). Identification of three new alternatively spliced variants of the rat mu opioid

receptor gene: dissociation of affinity and efficacy. J Neurochem 91, 881-890.

Pasternak, G.W. (2004). Multiple opiate receptors: deja vu all over again.

Neuropharmacology 47 Suppl 1, 312-323.

Pasternak, G.W. (2005). Molecular biology of opioid analgesia. J Pain Symptom Manage

29, S2-9.

Patel, M.B., Patel, C.N., Rajashekara, V., and Yoburn, B.C. (2002). Opioid agonists

differentially regulate mu-opioid receptors and trafficking proteins in vivo. Mol

Pharmacol 62, 1464-1470.

Peretti, D., Dahan, N., Shimoni, E., Hirschberg, K., and Lev, S. (2008). Coordinated lipid

transfer between the endoplasmic reticulum and the Golgi complex requires the VAP

proteins and is essential for Golgi-mediated transport. Mol Biol Cell 19, 3871-3884.

Petaja-Repo, U.E., Hogue, M., Laperriere, A., Bhalla, S., Walker, P., and Bouvier, M.

(2001). Newly synthesized human delta opioid receptors retained in the endoplasmic

reticulum are retrotranslocated to the cytosol, deglycosylated, ubiquitinated, and

degraded by the proteasome. J Biol Chem 276, 4416-4423.

Petaja-Repo, U.E., Hogue, M., Laperriere, A., Walker, P., and Bouvier, M. (2000).

Export from the endoplasmic reticulum represents the limiting step in the maturation and

cell surface expression of the human delta opioid receptor. J Biol Chem 275, 13727-

13736.

Petruzzi, R., Ferraro, T.N., Kurschner, V.C., Golden, G.T., and Berrettini, W.H. (1997).

The effects of repeated morphine exposure on mu opioid receptor number and affinity in

C57BL/6J and DBA/2J mice. Life Sci 61, 2057-2064.

Pezet, S., and McMahon, S.B. (2006). Neurotrophins: mediators and modulators of pain.

Annu Rev Neurosci 29, 507-538.

Pfeiffer, M., Kirscht, S., Stumm, R., Koch, T., Wu, D., Laugsch, M., Schroder, H., Hollt,

V., and Schulz, S. (2003). Heterodimerization of substance P and mu-opioid receptors

regulates receptor trafficking and resensitization. J Biol Chem 278, 51630-51637.

Pfeiffer, M., Koch, T., Schroder, H., Laugsch, M., Hollt, V., and Schulz, S. (2002).

Heterodimerization of somatostatin and opioid receptors cross-modulates

phosphorylation, internalization, and desensitization. J Biol Chem 277, 19762-19772.

159

Picetti, R., Caccavo, J.A., Ho, A., and Kreek, M.J. (2012). Dose escalation and dose

preference in extended-access heroin self-administration in Lewis and Fischer rats.

Psychopharmacology (Berl) 220, 163-172.

Picker, M.J., Negus, S.S., and Powell, K.R. (1991). Differential cross-tolerance to mu and

kappa opioid agonists in morphine-tolerant rats responding under a schedule of food

presentation. Psychopharmacology (Berl) 103, 129-135.

Polekhina, G., House, C.M., Traficante, N., Mackay, J.P., Relaix, F., Sassoon, D.A.,

Parker, M.W., and Bowtell, D.D. (2002). Siah ubiquitin ligase is structurally related to

TRAF and modulates TNF-alpha signaling. Nat Struct Biol 9, 68-75.

Prather, P.L., Tsai, A.W., and Law, P.Y. (1994). Mu and delta opioid receptor

desensitization in undifferentiated human neuroblastoma SHSY5Y cells. J Pharmacol

Exp Ther 270, 177-184.

Raehal, K.M., and Bohn, L.M. (2005). Mu opioid receptor regulation and opiate

responsiveness. AAPS J 7, E587-591.

Ren, K., and Dubner, R. (2007). Pain facilitation and activity-dependent plasticity in pain

modulatory circuitry: role of BDNF-TrkB signaling and NMDA receptors. Mol

Neurobiol 35, 224-235.

Reyes-Gibby, C.C., Shete, S., Rakvag, T., Bhat, S.V., Skorpen, F., Bruera, E., Kaasa, S.,

and Klepstad, P. (2007). Exploring joint effects of genes and the clinical efficacy of

morphine for cancer pain: OPRM1 and COMT gene. Pain 130, 25-30.

Ring, R.H., Alder, J., Fennell, M., Kouranova, E., Black, I.B., and Thakker-Varia, S.

(2006). Transcriptional profiling of brain-derived-neurotrophic factor-induced neuronal

plasticity: a novel role for nociceptin in hippocampal neurite outgrowth. J Neurobiol 66,

361-377.

Rios, C., Gomes, I., and Devi, L.A. (2006). mu opioid and CB1 cannabinoid receptor

interactions: reciprocal inhibition of receptor signaling and neuritogenesis. Br J

Pharmacol 148, 387-395.

Roberts, A.J., Gold, L.H., Polis, I., McDonald, J.S., Filliol, D., Kieffer, B.L., and Koob,

G.F. (2001). Increased ethanol self-administration in delta-opioid receptor knockout

mice. Alcohol Clin Exp Res 25, 1249-1256.

Roberts, A.J., McDonald, J.S., Heyser, C.J., Kieffer, B.L., Matthes, H.W., Koob, G.F.,

and Gold, L.H. (2000). mu-Opioid receptor knockout mice do not self-administer alcohol.

J Pharmacol Exp Ther 293, 1002-1008.

Robinson, T.E., and Kolb, B. (1999). Morphine alters the structure of neurons in the

nucleus accumbens and neocortex of rats. Synapse 33, 160-162.

160

Robinson, T.E., and Kolb, B. (2004). Structural plasticity associated with exposure to

drugs of abuse. Neuropharmacology 47 Suppl 1, 33-46.

Rossi, G.C., Brown, G.P., Leventhal, L., Yang, K., and Pasternak, G.W. (1996). Novel

receptor mechanisms for heroin and morphine-6 beta-glucuronide analgesia. Neurosci

Lett 216, 1-4.

Roy, S., Liu, H.C., and Loh, H.H. (1998). mu-Opioid receptor-knockout mice: the role of

mu-opioid receptor in gastrointestinal transit. Brain Res Mol Brain Res 56, 281-283.

Roy, S., Wang, J.H., Balasubramanian, S., Sumandeep, Charboneau, R., Barke, R., and

Loh, H.H. (2001). Role of hypothalamic-pituitary axis in morphine-induced alteration in

thymic cell distribution using mu-opioid receptor knockout mice. J Neuroimmunol 116,

147-155.

Rozenfeld, R., and Devi, L.A. (2007). Receptor heterodimerization leads to a switch in

signaling: beta-arrestin2-mediated ERK activation by mu-delta opioid receptor

heterodimers. FASEB J 21, 2455-2465.

Russo, S.J., Dietz, D.M., Dumitriu, D., Morrison, J.H., Malenka, R.C., and Nestler, E.J.

(2010). The addicted synapse: mechanisms of synaptic and structural plasticity in nucleus

accumbens. Trends Neurosci 33, 267-276.

Salinas, P.C., and Zou, Y. (2008). Wnt signaling in neural circuit assembly. Annu Rev

Neurosci 31, 339-358.

Schnell, S.A., and Wessendorf, M.W. (2004). Expression of MOR1C-like mu-opioid

receptor mRNA in rats. J Comp Neurol 473, 213-232.

Schubert, U., Anton, L.C., Gibbs, J., Norbury, C.C., Yewdell, J.W., and Bennink, J.R.

(2000). Rapid degradation of a large fraction of newly synthesized proteins by

proteasomes. Nature 404, 770-774.

Schulz, S., Mayer, D., Pfeiffer, M., Stumm, R., Koch, T., and Hollt, V. (2004). Morphine

induces terminal micro-opioid receptor desensitization by sustained phosphorylation of

serine-375. EMBO J 23, 3282-3289.

Shabalina, S.A., Zaykin, D.V., Gris, P., Ogurtsov, A.Y., Gauthier, J., Shibata, K.,

Tchivileva, I.E., Belfer, I., Mishra, B., Kiselycznyk, C., et al. (2009). Expansion of the

human mu-opioid receptor gene architecture: novel functional variants. Hum Mol Genet

18, 1037-1051.

Sharma, S.K., Klee, W.A., and Nirenberg, M. (1975). Dual regulation of adenylate

cyclase accounts for narcotic dependence and tolerance. Proc Natl Acad Sci U S A 72,

3092-3096.

161

Shi, J., Hui, L., Xu, Y., Wang, F., Huang, W., and Hu, G. (2002). Sequence variations in

the mu-opioid receptor gene (OPRM1) associated with human addiction to heroin. Hum

Mutat 19, 459-460.

Shi, L., Yue, J., You, Y., Yin, B., Gong, Y., Xu, C., Qiang, B., Yuan, J., Liu, Y., and

Peng, X. (2006). Dok5 is substrate of TrkB and TrkC receptors and involved in

neurotrophin induced MAPK activation. Cell Signal 18, 1995-2003.

Shmueli, A., Tsai, Y.C., Yang, M., Braun, M.A., and Weissman, A.M. (2009). Targeting

of gp78 for ubiquitin-mediated proteasomal degradation by Hrd1: cross-talk between E3s

in the endoplasmic reticulum. Biochem Biophys Res Commun 390, 758-762.

Sia, A.T., Lim, Y., Lim, E.C., Goh, R.W., Law, H.Y., Landau, R., Teo, Y.Y., and Tan,

E.C. (2008). A118G single nucleotide polymorphism of human mu-opioid receptor gene

influences pain perception and patient-controlled intravenous morphine consumption

after intrathecal morphine for postcesarean analgesia. Anesthesiology 109, 520-526.

Simon, E.J., Hiller, J.M., and Edelman, I. (1973). Stereospecific binding of the potent

narcotic analgesic (3H) Etorphine to rat-brain homogenate. Proc Natl Acad Sci U S A 70,

1947-1949.

Simonin, F., Valverde, O., Smadja, C., Slowe, S., Kitchen, I., Dierich, A., Le Meur, M.,

Roques, B.P., Maldonado, R., and Kieffer, B.L. (1998). Disruption of the kappa-opioid

receptor gene in mice enhances sensitivity to chemical visceral pain, impairs

pharmacological actions of the selective kappa-agonist U-50,488H and attenuates

morphine withdrawal. EMBO J 17, 886-897.

Smith, M.H., Ploegh, H.L., and Weissman, J.S. (2011). Road to ruin: targeting proteins

for degradation in the endoplasmic reticulum. Science 334, 1086-1090.

Snyder, S.H. (2004). Opiate receptors and beyond: 30 years of neural signaling research.

Neuropharmacology 47 Suppl 1, 274-285.

Snyder, S.H., and Childers, S.R. (1979). Opiate receptors and opioid peptides. Annu Rev

Neurosci 2, 35-64.

Sokolov, B.P., Jiang, L., Trivedi, N.S., and Aston, C. (2003). Transcription profiling

reveals mitochondrial, ubiquitin and signaling systems abnormalities in postmortem

brains from subjects with a history of alcohol abuse or dependence. J Neurosci Res 72,

756-767.

Somogyi, A.A., Barratt, D.T., and Coller, J.K. (2007). Pharmacogenetics of opioids. Clin

Pharmacol Ther 81, 429-444.

162

Sora, I., Elmer, G., Funada, M., Pieper, J., Li, X.F., Hall, F.S., and Uhl, G.R. (2001). Mu

opiate receptor gene dose effects on different morphine actions: evidence for differential

in vivo mu receptor reserve. Neuropsychopharmacology 25, 41-54.

Sora, I., Takahashi, N., Funada, M., Ujike, H., Revay, R.S., Donovan, D.M., Miner, L.L.,

and Uhl, G.R. (1997). Opiate receptor knockout mice define mu receptor roles in

endogenous nociceptive responses and morphine-induced analgesia. Proc Natl Acad Sci

U S A 94, 1544-1549.

Stafford, K., Gomes, A.B., Shen, J., and Yoburn, B.C. (2001). mu-Opioid receptor

downregulation contributes to opioid tolerance in vivo. Pharmacol Biochem Behav 69,

233-237.

Stagljar, I., Korostensky, C., Johnsson, N., and te Heesen, S. (1998). A genetic system

based on split-ubiquitin for the analysis of interactions between membrane proteins in

vivo. Proc Natl Acad Sci U S A 95, 5187-5192.

Studier, F.W. (2005). Protein production by auto-induction in high density shaking

cultures. Protein Expr Purif 41, 207-234.

Suzuki, T., Otani, K., Koike, Y., and Misawa, M. (1988). Genetic differences in

preferences for morphine and codeine in Lewis and Fischer 344 inbred rat strains. Jpn J

Pharmacol 47, 425-431.

Szargel, R., Rott, R., Eyal, A., Haskin, J., Shani, V., Balan, L., Wolosker, H., and

Engelender, S. (2009). Synphilin-1A inhibits seven in absentia homolog (SIAH) and

modulates alpha-synuclein monoubiquitylation and inclusion formation. J Biol Chem

284, 11706-11716.

Szeto, C.Y., Tang, N.L., Lee, D.T., and Stadlin, A. (2001). Association between mu

opioid receptor gene polymorphisms and Chinese heroin addicts. Neuroreport 12, 1103-

1106.

Talbot, J.N., Skifter, D.A., Bianchi, E., Monaghan, D.T., Toews, M.L., and Murrin, L.C.

(2009). Regulation of mu opioid receptor internalization by the scaffold protein

RanBPM. Neurosci Lett 466, 154-158.

Tan, E.C., Tan, C.H., Karupathivan, U., and Yap, E.P. (2003). Mu opioid receptor gene

polymorphisms and heroin dependence in Asian populations. Neuroreport 14, 569-572.

Tang, X.C., McFarland, K., Cagle, S., and Kalivas, P.W. (2005). Cocaine-induced

reinstatement requires endogenous stimulation of mu-opioid receptors in the ventral

pallidum. J Neurosci 25, 4512-4520.

163

Tanowitz, M., and von Zastrow, M. (2003). A novel endocytic recycling signal that

distinguishes the membrane trafficking of naturally occurring opioid receptors. J Biol

Chem 278, 45978-45986.

Tegeder, I., and Geisslinger, G. (2004). Opioids as modulators of cell death and survival-

-unraveling mechanisms and revealing new indications. Pharmacol Rev 56, 351-369.

Terenius, L. (1973). Stereospecific interaction between narcotic analgesics and a synaptic

plasm a membrane fraction of rat cerebral cortex. Acta Pharmacol Toxicol (Copenh) 32,

317-320.

Trejo, J., Hammes, S.R., and Coughlin, S.R. (1998). Termination of signaling by

protease-activated receptor-1 is linked to lysosomal sorting. Proc Natl Acad Sci U S A

95, 13698-13702.

Trescot, A.M., Datta, S., Lee, M., and Hansen, H. (2008). Opioid pharmacology. Pain

Physician 11, S133-153.

Trigo, J.M., Martin-Garcia, E., Berrendero, F., Robledo, P., and Maldonado, R. (2010).

The endogenous opioid system: a common substrate in drug addiction. Drug Alcohol

Depend 108, 183-194.

Tsai, Y.C., and Weissman, A.M. (2011). Ubiquitylation in ERAD: reversing to go

forward? PLoS Biol 9, e1001038.

Tuoc, T.C., and Stoykova, A. (2010). Roles of the ubiquitin-proteosome system in

neurogenesis. Cell Cycle 9, 3174-3180.

Tzschentke, T.M. (1998). Measuring reward with the conditioned place preference

paradigm: a comprehensive review of drug effects, recent progress and new issues. Prog

Neurobiol 56, 613-672.

Tzschentke, T.M. (2007). Measuring reward with the conditioned place preference (CPP)

paradigm: update of the last decade. Addict Biol 12, 227-462.

Uchida, M., Enomoto, A., Fukuda, T., Kurokawa, K., Maeda, K., Kodama, Y., Asai, N.,

Hasegawa, T., Shimono, Y., Jijiwa, M., et al. (2006). Dok-4 regulates GDNF-dependent

neurite outgrowth through downstream activation of Rap1 and mitogen-activated protein

kinase. J Cell Sci 119, 3067-3077.

Ueda, H., Harada, H., Nozaki, M., Katada, T., Ui, M., Satoh, M., and Takagi, H. (1988).

Reconstitution of rat brain mu opioid receptors with purified guanine nucleotide-binding

regulatory proteins, Gi and Go. Proc Natl Acad Sci U S A 85, 7013-7017.

van Ree, J.M., and de Wied, D. (1980). Involvement of neurohypophyseal peptides in

drug-mediated adaptive responses. Pharmacol Biochem Behav 13 Suppl 1, 257-263.

164

Volkow, N.D., and Li, T.K. (2005). Drugs and alcohol: treating and preventing abuse,

addiction and their medical consequences. Pharmacol Ther 108, 3-17.

von Zastrow, M., Svingos, A., Haberstock-Debic, H., and Evans, C. (2003). Regulated

endocytosis of opioid receptors: cellular mechanisms and proposed roles in physiological

adaptation to opiate drugs. Curr Opin Neurobiol 13, 348-353.

Waldhoer, M., Bartlett, S.E., and Whistler, J.L. (2004). Opioid receptors. Annu Rev

Biochem 73, 953-990.

Wang, D., Quillan, J.M., Winans, K., Lucas, J.L., and Sadee, W. (2001). Single

nucleotide polymorphisms in the human mu opioid receptor gene alter basal G protein

coupling and calmodulin binding. J Biol Chem 276, 34624-34630.

Wang, D., Sadee, W., and Quillan, J.M. (1999). Calmodulin binding to G protein-

coupling domain of opioid receptors. J Biol Chem 274, 22081-22088.

Wang, H.L., Hsu, C.Y., Huang, P.C., Kuo, Y.L., Li, A.H., Yeh, T.H., Tso, A.S., and

Chen, Y.L. (2005). Heterodimerization of opioid receptor-like 1 and mu-opioid receptors

impairs the potency of micro receptor agonist. J Neurochem 92, 1285-1294.

Wang, J.B., Johnson, P.S., Persico, A.M., Hawkins, A.L., Griffin, C.A., and Uhl, G.R.

(1994). Human mu opiate receptor. cDNA and genomic clones, pharmacologic

characterization and chromosomal assignment. FEBS Lett 338, 217-222.

Wheeler, T.C., Chin, L.S., Li, Y., Roudabush, F.L., and Li, L. (2002). Regulation of

synaptophysin degradation by mammalian homologues of seven in absentia. J Biol Chem

277, 10273-10282.

Whistler, J.L., Chuang, H.H., Chu, P., Jan, L.Y., and von Zastrow, M. (1999). Functional

dissociation of mu opioid receptor signaling and endocytosis: implications for the biology

of opiate tolerance and addiction. Neuron 23, 737-746.

Williams, J.T., Christie, M.J., and Manzoni, O. (2001). Cellular and synaptic adaptations

mediating opioid dependence. Physiol Rev 81, 299-343.

Wong, Y.H., Demoliou-Mason, C.D., and Barnard, E.A. (1988). ADP-ribosylation with

pertussis toxin modulates the GTP-sensitive opioid ligand binding in digitonin-soluble

extracts of rat brain membranes. J Neurochem 51, 114-121.

Wu, D.F., Koch, T., Liang, Y.J., Stumm, R., Schulz, S., Schroder, H., and Hollt, V.

(2007). Membrane glycoprotein M6a interacts with the micro-opioid receptor and

facilitates receptor endocytosis and recycling. J Biol Chem 282, 22239-22247.

165

Wyles, J.P., McMaster, C.R., and Ridgway, N.D. (2002). Vesicle-associated membrane

protein-associated protein-A (VAP-A) interacts with the oxysterol-binding protein to

modify export from the endoplasmic reticulum. J Biol Chem 277, 29908-29918.

Xu, J., Xu, M., Hurd, Y.L., Pasternak, G.W., and Pan, Y.X. (2009). Isolation and

characterization of new exon 11-associated N-terminal splice variants of the human mu

opioid receptor gene. J Neurochem 108, 962-972.

Xue, J.C., Chen, C., Zhu, J., Kunapuli, S., DeRiel, J.K., Yu, L., and Liu-Chen, L.Y.

(1994). Differential binding domains of peptide and non-peptide ligands in the cloned rat

kappa opioid receptor. J Biol Chem 269, 30195-30199.

Yang, L., Wang, S., Lim, G., Sung, B., Zeng, Q., and Mao, J. (2008). Inhibition of the

ubiquitin-proteasome activity prevents glutamate transporter degradation and morphine

tolerance. Pain 140, 472-478.

Yoshida, Y., Chiba, T., Tokunaga, F., Kawasaki, H., Iwai, K., Suzuki, T., Ito, Y.,

Matsuoka, K., Yoshida, M., Tanaka, K., et al. (2002). E3 ubiquitin ligase that recognizes

sugar chains. Nature 418, 438-442.

Yun, S., Moller, A., Chae, S.K., Hong, W.P., Bae, Y.J., Bowtell, D.D., Ryu, S.H., and

Suh, P.G. (2008). Siah proteins induce the epidermal growth factor-dependent

degradation of phospholipase Cepsilon. J Biol Chem 283, 1034-1042.

Zangen, A., Ikemoto, S., Zadina, J.E., and Wise, R.A. (2002). Rewarding and

psychomotor stimulant effects of endomorphin-1: anteroposterior differences within the

ventral tegmental area and lack of effect in nucleus accumbens. J Neurosci 22, 7225-

7233.

Zhu, J., Chen, C., Xue, J.C., Kunapuli, S., DeRiel, J.K., and Liu-Chen, L.Y. (1995).

Cloning of a human kappa opioid receptor from the brain. Life Sci 56, PL201-207.

Zhu, Y., King, M.A., Schuller, A.G., Nitsche, J.F., Reidl, M., Elde, R.P., Unterwald, E.,

Pasternak, G.W., and Pintar, J.E. (1999). Retention of supraspinal delta-like analgesia

and loss of morphine tolerance in delta opioid receptor knockout mice. Neuron 24, 243-

252.

VITA Stephanie Justice-Bitner

EDUCATIONAL BACKGROUND

Master of Science in Veterinary Science, 2004

University of Nebraska, Lincoln, NE

Non-thesis, emphasis in immunology

Bachelor of Science in Microbiology, 1997

Colorado State University, Fort Collins, CO

PROFESSIONAL EXPERIENCE

Visiting Assistant Professor of Biology, Department of Biology 8/11-present

King’s College, Wilkes-Barre, PA

Adjunct Professor of Biology, Department of Biology 8/09-5-09

Elizabethtown College, Elizabethtown, PA

Temporary Faculty- Biology, Kulpmont/Berwick 8/04-12/05

Luzerne County Community College, Nanticoke, PA

Research Tech III, Veterinary Diagnostic Center 8/03-1/04

University of Nebraska, Lincoln, NE

PUBLICATIONS

Justice-Bitner S, Petko, J, Jin J, Wong V, Kittanakom S, Ferraro T, Stagljar I, and

Levenson R, "MOR is Not Enough: Identification of Novel mu-Opioid Receptor

Interacting Proteins Using Traditional and Modified Membrane Yeast Two-

Hybrid Screens" (Submitted)

ABSTRACTS

“Discovery of Novel mu-Opioid Interacting Proteins Involved in the Ubiquitin

Pathway.”

Ubiquitin Drug Discovery and Diagnostics Conference, Hyatt at the Bellevue,

Philadelphia, PA, October 2009.