NEUROPROTECTIVE SIGNALING THROUGH METABOTROPIC GLUTAMATE

RECEPTOR 1

A Dissertation

submitted to the Faculty of the

Graduate School of Arts and Sciences

of Georgetown University

in partial fulfillment of the requirements for the

degree of

Doctor of Philosophy

in Neuroscience

By

Andrew Clayton Emery, M.A.

Washington, DC

April 12, 2011

ii

Copyright 2011 by Andrew Clayton Emery

All Rights Reserved

iii

NEUROPROTECTIVE SIGNALING THROUGH METABOTROPIC GLUTAMATE

RECEPTOR 1

Andrew C. Emery, M.A.

Thesis Advisor: Jarda T. Wroblewski, Ph.D.

ABSTRACT

Metabotropic glutamate receptor 1 (mGlu1) is a G protein-coupled receptor which enhances

the hydrolysis of membrane phosphoinositides (PI). Additionally, mGlu1 receptors have been

shown to stimulate cytoprotective signaling in the presence of the endogenous ligand glutamate.

This goal of the experiments in the following thesis was to (1) characterize the signal

transduction pathway through which mGlu1 receptors protect cells expressing these receptors

from toxicity. Instead of the classical G protein-mediated (PI) signaling, the following

experiments indicate that mGlu1a receptors cause a sustained phosphorylation of ERK that is -

arrestin-1 dependent. This G protein-independent signaling pathway was necessary for protective

signaling and was stimulated by glutamate, but not quisqualate, demonstrating ligand bias at this

receptor. (2) Therefore, pharmacological and mutational studies were carried out to investigate

the molecular basis of this ligand bias. Together, these studies indicate the existence of 3 classes

of mGlu1a receptor agonists: (a) unbiased agonists, such as glutamate, which stimulate both

signal transduction pathways, (b) biased agonists, such as quisqualate, which only stimulate PI

hydrolysis, and (c) agonists which are biased toward sustained ERK phosphorylation and

protective signaling. In these studies, glutaric and succinic acid were identified as protection-

biased mGlu1a receptor agonists. Further pharmacological studies indicate that quisqualate does

not inhibit glutamate-induced protection and glutaric acid fails to inhibit glutamate-induced PI

iv

hydrolysis, suggesting the existence of two distinct, non-overlapping agonist binding sites on

mGlu1a receptors.

v

I wish to extend my sincere gratitude to my family and colleagues who have been so helpful and

supportive throughout the process of this thesis. In particular I wish to thank my wife, Maria Jena

Emery, for all of her love and care, as well as my parents, Clay and Wendy Emery. My advisor,

Dr. Jarda Wroblewski, has served as a kindly and sage mentor throughout my entire tenure in his

laboratory and was instrumental through the entire process of this thesis. Many of the

experiments performed herein were successful due to the assistance of Dr. Sergey Pshenichkin,

Ewa Grajkowska, and Rodrigue Takoudjou, S.J. Furthermore, Drs. Barry Wolfe and Bob Yasuda

were extremely helpful in regular discussion of experimental methods and results. I remain

especially grateful to Dr. Jean Wrathall, who provided grant support to me from the Training

Program in Neural Injury and Recovery for the entirety of my thesis research.

Many thanks,

Andrew C. Emery

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TABLE OF CONTENTS

ABSTRACT .................................................................................................................... iii

ACKNOLEDGEMENTS ................................................................................................. v

TABLE OF CONTENTS ................................................................................................ vi

LIST OF FIGURES ....................................................................................................... vii

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

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

CHAPTER 1: Thesis Introduction .................................................................................... 1

MATERIALS AND METHODS ..................................................................................... 9

CHAPTER 2: The Protective Signaling of Metabotropic Glutamate Receptor 1 is Mediated by

Sustained β-Arrestin-1-dependent ERK Phosphorylation .............................................. 14

INTRODUCTION ......................................................................................................... 15

RESULTS ...................................................................................................................... 17

DISCUSSION ................................................................................................................. 47

CHAPTER 2: Metabotropic Glutamate Receptor 1 Possesses Two Glutamate Binding Sites

Coupled to Different Signal Transduction Systems........................................................ 50

INTRODUCTION .......................................................................................................... 51

RESULTS ....................................................................................................................... 54

DISCUSSION ................................................................................................................. 88

CHAPTER 4: Thesis Conclusion.................................................................................... 94

REFERENCES ............................................................................................................. 100

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LIST OF FIGURES

Figure 1. Time course of protection by glutamate in CHO cells expressing mGlu1a receptors

........................................................................................................................................ 18

Figure 2. Efficacy of glutamate to protect CHO cells .................................................... 19

Figure 3. Potency of glutamate to protect CHO cells expressing mGlu1a receptors ..... 21

Figure 4. Inhibition of the protective effect of glutamate by mGlu1-selective noncompetitive

antagonists....................................................................................................................... 22

Figure 5. Noncompetitive inhibition of protection by glutamate ................................... 23

Figure 6. Pharmacology of PI hydrolysis through mGlu1a receptors ............................ 25

Figure 7. Bioassay demonstrating the stability of glutamate and quisqualate ................ 27

Figure 8. Receptor reserve for mGlu1a receptor-mediated PI hydrolysis ...................... 28

Figure 9. Glutamate-induced protection is PLC-independent but MEK and dynamin-dependent

......................................................................................................................................... 30

Figure 10. Internalization of mGlu1a receptors is dynamin-dependent ......................... 32

Figure 11. Glutamate-induced protection is PLC-independent but MEK and dynamin-dependent

in neurons ........................................................................................................................ 33

Figure 12. ERK phosphorylation due to mGlu1 agonists .............................................. 35

Figure 13. ELISA-based quantification of phosphorylated ERK ................................... 36

Figure 14. Agonist profiles of ERK phosphorylation through mGlu1a receptors .......... 37

Figure 15. Silencing of -arrestin-1 with shRNA ........................................................... 40

Figure 16. Knockdown β-arrestin-1 of does not impair classical mGlu1a receptor signaling

......................................................................................................................................... 41

Figure 17. Protective signaling through mGlu1a is abolished by shRNA silencing of β-arrestin-1

......................................................................................................................................... 42

Figure 18. Endocytosis of mGlu1a receptors is β-arrestin-1-dependent......................... 44

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Figure 19. Sustained ERK phosphorylation through mGlu1a receptors is β-arrestin-1-dependent

......................................................................................................................................... 45

Figure 20. Schematic representation of signal transduction through mGlu1 receptors . 46

Figure 21. Agonist profile for mGlu1a-mediated PI hydrolysis .................................... 56

Figure 22. Agonist profile for mGlu1a-mediated transient ERK phosphorylation ....... 57

Figure 23. Agonist profile for mGlu1a-mediated sustained ERK phosphorylation

......................................................................................................................................... 58

Figure 24. Agonist profile for mGlu1a-mediated protective signaling.......................... 60

Figure 25. Competitive antagonism of glutamate-induced PI hydrolysis by LY367685

......................................................................................................................................... 62

Figure 26. Competitive antagonism of glutamate-induced PI hydrolysis by 3-MATIDA

......................................................................................................................................... 63

Figure 27. Competitive antagonism of glutamate-induced transient ERK phosphorylation

......................................................................................................................................... 64

Figure 28. Lack of competitive antagonism on glutamate-induced sustained ERK

phosphorylation.............................................................................................................. 66

Figure 29. Lack of competitive antagonism on glutamate-induced protective signaling

......................................................................................................................................... 67

Figure 30. Protein expression levels of mutant mGlu1a receptors ................................. 69

Figure 31. Lack of classical mGlu1a receptor signaling in T188A mutant receptors .... 70

Figure 32. Silencing of -arrestin-1 in CHO cells expressing T188A mutant mGlu1a receptors

......................................................................................................................................... 71

Figure 33. -arrestin-1 dependent sustained ERK phosphorylation in T188A mutant mGlu1a

receptors .......................................................................................................................... 72

Figure 34. Glutamate-induced protective signaling in T188A mutant mGlu1a receptors

......................................................................................................................................... 73

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Figure 35. Classical signaling in R323V and K409A mutant mGlu1a receptors .......... 75

Figure 36. ERK phosphorylation is G protein-dependent in R323V and K409A mutant mGlu1a

receptors .......................................................................................................................... 76

Figure 37. R323V and K409A mutant mGlu1a receptors do not stimulate long-term ERK

phosphorylation............................................................................................................... 77

Figure 38. R323V and K409A mutant mGlu1a receptors do not protect CHO cells ..... 78

Figure 39. Effects of glutaric and succinic acids on PI hydrolysis ................................. 80

Figure 40. Glutaric acid is an mGlu1a receptor agonist that causes ERK phosphorylation

......................................................................................................................................... 81

Figure 41. Glutaric acid protects CHO cells through activity at mGlu1a receptors ....... 82

Figure 42. Glutaric acid protects cerebellar granule neurons through mGlu1a receptors

......................................................................................................................................... 83

Figure 43. Succinic acid protects CHO cells through activity at mGlu1a receptors ...... 84

Figure 44. Quisqualate fails to inhibit glutamate-induced protection at mGlu1a receptors

......................................................................................................................................... 86

Figure 45. Glutaric acid fails to inhibit glutamate-induced PI hydrolysis at mGlu1a receptors

......................................................................................................................................... 87

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LIST OF TABLES

Table 1. Summary of mGlu1a receptor pharmacology ................................................... 24

Table 2. Different responses induced by three classes of agonists acting at wild type and mutated

mGlu1a receptors ........................................................................................................... 90

Table 3.Sequences of PCR primers for creating mutant mGlu1 receptors ................... 99

xi

LIST OF ABBREVIATIONS

Asp, L-aspartate; CHO, Chinese hamster ovary; CPCCOEt, 7-hydroxyiminocyclopropan[b]

chromen-1a-carboxylic acid ethyl ester; DHPG, (S)-3,5-Dihydroxyphenylglycine; DTT,

dithiothreitol; ELISA, Enzyme-linked immunosorbent assay; ERK, extracellular signal-regulated

kinase; GA, L-glutaric acid; Glu, L-glutamate; GPCR, G protein-coupled receptor; IP, inositol

phosphate; JNJ16259685, (3,4-dihydro-2H-pyrano[2,3]b quinolin-7-yl) (cis-4-

methoxycyclohexyl) methanone; L-CA, L-cysteic acid; LY367385, (+)-2-Methyl-4-

carboxyphenyl glycine; MAPK, mitogen-activated protein kinase; MEK, ERK kinase; mGlu,

metabotropic glutamate; MTT, [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium

Bromide]; PI, phosphatidylinositdes; PKC, Protein kinase C; PLC, Phospholipase C; Quis, L-

quisqualate; TPA, 12-O-tetradecanoylphorbol-13-acetate; YM 298198, 6-Amino-N-cyclohexyl-

N,3-dimethylthiazolo[3,2-a]benzimidazole-2-carboxamide hydrochloride.

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CHAPTER 1

Thesis Introduction

2

INTRODUCTION

Properties of metabotropic glutamate receptors

Metabotropic glutamate (mGlu) receptors are a family of G protein-coupled receptors linked

to multiple second messenger systems (Conn and Pin, 1997). Eight mGlu receptors have been

cloned and have been categorized into three groups based on sequence homology and

pharmacology (Conn and Pin, 1997; Pin and Duvoisin, 1995). Group I includes mGlu1 and

mGlu5, group II mGlu2 and mGlu3, and group III mGlu4, mGlu6, mGlu7, and mGlu8. These

receptors form family C of G protein coupled receptors, which also includes the parathyroid

calcium-sensing receptor (Garrett et al., 1995), the GABAB receptor (Kaupmann et al., 1997),

and pheromone receptors (Matsunami and Buck, 1997). Structural features of this family of

receptors include a large extracellular domain containing an agonist binding site (Takahashi et

al., 1993), seven transmembrane spanning domains, and a variable-length intracellular C-

terminal domain (Bhave et al., 2003). The second intracellular loop and portions of the C-

terminus are responsible for binding of G proteins and therefore for the coupling of mGlu

receptors to the different second messenger systems (Gomeza et al., 1996a; Pin et al., 1994).

Initial evidence for the existence of mGlu1 receptors came from reports which described the

ability of glutamate to stimulate the accumulation of inositol phosphates in cultured striatal and

cerebellar granule neurons (Nicoletti et al., 1986; Sladeczek et al., 1985). Two laboratories later

cloned these receptors (Houamed et al., 1991; Masu et al., 1991) and further studies

demonstrated that group I mGlu receptors stimulate phospholipase C (PLC) via coupling to

Gq/11 (Aramori and Nakanishi, 1992; Pickering et al., 1993). Activated PLC cleaves

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phosphatidylinositol-4,5-bisphosphate into inositol-1,4,5-trisphosphate and diacylglycerol.

Inositol phosphates then cause calcium release from intracellular stores, which happens due to

activation of both IP3 receptors and ryanodine receptors, both of which are located on the surface

of the endoplasmic reticulum (del Rio et al., 1999). Released calcium and diacylglycerol cause

the phosphorylation of protein kinase C (Nishizuka, 1995). Furthermore, agonist stimulation of

group I mGlu receptors has more recently been shown to cause a transient protein kinase C-

dependent phosphorylation of extracellular signal-regulated kinase (ERK) (Choe and Wang,

2001; Ferraguti et al., 1999; Karim et al., 2001).

Physiological roles of mGlu1 receptors

Metabotropic glutamate receptor 1 is highly expressed in the granule and Purkinje cells of the

cerebellum, in the substania nigra pars compacta, in the septal nuclei, in the relay nuclei of the

dorsal thalamus, and throughout the dorsal pallidum (Martin et al., 1992). Studies of the

localization of mGlu1 receptors have indicated that they are most highly-expressed on

postsynaptic membranes at excitatory synapses (Baude et al., 1993). Most physiological studies

on mGlu1 receptors have been conducted to investigate its roles in cerebellar long-term

depression at the parallel fiber-Purkinje cell synapse (Kano et al., 2008). Knockout mice lacking

the gene (GRM1) for mGlu1 receptors are ataxic and have impaired motor learning skills and

cerebellar LTD (Aiba et al., 1994). Reintroduction of mGlu1 receptors in these mice corrects

ataxia and aberrant cerebellar LTD (Ichise et al., 2000). Additionally, mGlu1 receptors seem to

play a role in the synaptogenesis of the cerebellar cortex since GRM1 knockout mice have

impaired synapse elimination at parallel fiber-Purkinje cell synapses (Kano et al., 1997). In

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addition to its roles in synaptic plasticity, mGlu1 receptors have been shown to mediate slow

EPSPs at these synapses (Batchelor et al., 1997).

Pharmacology of Group I Metabotropic Glutamate Receptors

Pharmacologically, group I mGlu receptors can be distinguished from other groups of mGlu

receptors by the action of their most potent agonist quisqualate, and by the group I-selective

agonist 3,5-dihydroxyphenylglycine (DHPG) (Ito et al., 1992; Schoepp et al., 1994). The

nonselective agonist of mGlu receptors 1-Aminocyclopentane-trans-1,3-di

carboxylic acid (trans-ACPD) stimulates PI hydrolysis through mGlu1 receptors, however it is

less potent than the endogenous agonist glutamate (Thomsen et al., 1993). L-aspartate also acts

as an agonist at mGlu1 receptors, but it is also active at NMDA receptors (Chen et al., 2005),

which is probably responsible for limiting its use on mGlu1 receptors in the literature.

Additionally, sulfur-containing amino acids, such as L-cysteic acid and L-homocysteic acid have

been shown to possess the properties of agonists at mGlu1 receptors (Porter and Roberts, 1993;

Shi et al., 2003). Despite the relatively high number of available ligands for mGlu1 receptors, an

agonist which is selective for mGlu1 receptors and inactive at mGlu5 receptors has not yet been

identified.

Competitive antagonism of mGlu1 receptors was initially accomplished using several analogs

of phenylglycine, most notably MCPG (Eaton et al., 1993). None of these compounds are

selective for mGlu1 receptors (Hayashi et al., 1994), but further modifications led to the

development of LY367385, which is moderately potent and for mGlu1 receptors with little

action at mGlu5 receptors (Clark et al., 1997). Additionally, 3-MATIDA, a rigidified

5

phenylglycine analog, selectively competitively inhibits mGlu1 receptors (Moroni et al., 2002).

The most potent and selective antagonists at mGlu1 receptors are the noncompetitive

antagonists, which bind to an allosteric site situated on the seventh transmembrane domain of the

receptor (Litschig et al., 1999). A number of these compounds exist and are typified by the first

to be synthesized, CPCCOEt (Hermans et al., 1998). Subsequently, more selective and potent

noncompetitive antagonists which are systemically active have been discovered, and these

compounds include YM-298198 (Kohara et al., 2005), and JNJ 16259685 (Lavreysen et al.,

2004) .

Metabotropic Glutamate Receptors in Neuroprotection

Several studies indicate that activation of group I mGlu receptors promotes neuronal death.

Such results have been demonstrated in an in vivo model of rat traumatic brain injury and in an in

vitro model of traumatic injury of rat cortical neurons (Mukhin et al., 1996). The toxic effects of

group I mGlu receptors appear to be mediated through mechanisms including the activation of

protein kinase C (Bruno et al., 1995), potentiation of NMDA and AMPA currents (Aniksztejn et

al., 1992; Bleakman et al., 1992; Harvey and Collingridge, 1993; Meguro et al., 1992), and the

activation of voltage-gated calcium channels (Chavis et al., 1995). In contrast, multiple other

studies indicate that in the presence of glutamate, mGlu1 induces signaling that facilitates growth

and development as opposed to neurotoxicity. Activation of mGlu1 receptors has been shown to

increase tolerance to ischemia in the hippocampus, thus minimizing cellular damage due to

ischemia (Werner et al., 2007). When stimulated with glutamate, mGlu1 has been shown to

stimulate axon elongation in the developing central nervous system (Kreibich et al., 2004) and

6

outgrowth of dendritic spines in the developing hippocampus (Vanderklish and Edelman, 2002).

It has recently been described that mGlu1a produces dual neuroprotective and neurotoxic

signaling in cerebellar and cortical neurons (Pshenichkin et al., 2008). Thus, mGlu1a exhibits the

properties of a dependence receptor (Chao, 2003; Mehlen and Bredesen, 2004), inducing

apoptosis in the absence of glutamate, while promoting neuronal survival in its presence.

However, the molecular mechanisms of this neuroprotective signaling are unknown.

Regulation of mGlu1 Receptors by β-arrestin

Like other G protein-coupled receptors, desensitization and internalization of mGlu1 receptors

occurs both constitutively and in response to agonist stimulation. Initial studies on the

desensitization of mGlu1 receptors implicated protein kinase C (PKC), since phorbol esters

apparently increased desensitization of mGlu1 receptors and these increases were blocked by

inhibitors of PKC (Catania et al., 1991). Further studies on PKC regulation of mGlu1 receptors

indicate that PKC phosphorylates the receptor on the second intracellular loop (Alaluf et al.,

1995), which is where the G protein binds (Gomeza et al., 1996b), thus reducing G protein-

dependent activity through mGlu1 receptors. Along with PKC-mediated desensitization, in the

presence of an agonist, mGlu1 receptors undergo rapid homologous desensitization and are

endocytosed in a dynamin and β-arrestin-1-dependent manner (Mundell et al., 2001). During this

process, mGlu1 may be phosphorylated by several kinases. Homologous desensitization and

endocytosis likely require the activity of G-protein coupled receptor kinases (GRKs). Multiple

GRK isoforms, including GRK2, GRK4, GRK5, and GRK6 have been shown to have the

potential to phosphorylate mGlu1 (Dhami and Ferguson, 2006), however it remains unclear

7

which of these kinases phosphorylate native mGlu1 receptors. One study indicates that GRK2

regulates the desensitization of mGlu1 via a physical interaction with the second intracellular

loop (Dhami et al., 2004). However, phosphorylation of the C-terminus of mGlu1 receptors by

GRK4 appears to be necessary for β-arrestin1 binding (Iacovelli et al., 2003). Additionally, the

association of mGlu1 receptors with β-arrestin has been also shown to induce receptor-mediated

transient phosphorylation of ERK (Iacovelli et al., 2003).

Cellular Signaling through β-arrestin

ERK functions in the MAP kinase pathway downstream of MEK1/2 and the phosphorylation

of ERK is a critical step in signal transduction from the membrane to the nucleus and usually

causes protective or mitogenic cellular responses (Seger and Krebs, 1995). Although a relatively

recently described signal transduction pathway, numerous G protein-coupled receptors have been

shown to stimulate ERK phosphorylation in a β-arrestin-dependent manner (Kenakin, 2007).

Through numerous receptors, ERK can be activated by both β-arrestin dependent and G protein-

dependent signaling pathways (Wei et al., 2003). In several systems, including PAC1 and VPAC

receptors, these parallel pathways have been described where ERK phosphorylation due to G

protein activation is transient, whereas phosphorylation is sustained over time when ERK is

bound to β-arrestin (Broca et al., 2009). Transient, G protein-dependent ERK phosphorylation is

terminated by the action of β-arrestin, which is critical for the desensitization and internalization

of G protein-coupled receptors (Lohse et al., 1990).

Anti-apoptotic signaling through β-arrestin-dependent pathways has been demonstrated for

numerous G protein-coupled receptors. There is evidence that β-arrestin-mediated ERK

8

phosphorylation is independent from G protein signaling when β-arrestin is bound to a receptor.

Some receptors where this signaling mechanism has been characterized include the angiotensin

II type 1a receptor (Luttrell et al., 2001), the D3 dopamine receptor (Beom et al., 2004), the β2-

adrenergic receptor (Shenoy et al., 2006), and the mu opioid receptor (Macey et al., 2006). While

β-arrestin-mediated signaling has not previously been demonstrated due to mGlu1 receptor

activation, it is known that β-arrestin1 is involved in rapid agonist-induced internalization of

mGlu1 (Dale et al., 2001).

Ligand Bias

Through the same receptor, β-arrestin-mediated signaling may not follow an identical

pharmacological profile as classical G protein-mediated modes of signal transduction. This

phenomenon is known as ligand bias, which is a new and emerging concept in pharmacology

that has been shown to exist for multiple G protein-coupled receptors when various agonists

preferentially activate receptor conformations that are selectively conducive for different signal

transduction pathways (Violin and Lefkowitz, 2007). The earliest reported example of ligand

bias was in M1 muscarinic receptors, which activate both cAMP and PLC in response to their

endogenous agonist acetylcholine. However, several ligands were identified that selectively

activate PLC while blocking cAMP formation through these receptors (Fisher et al., 1993). Since

this initial report of ligand bias at different modes of G protein-mediated signaling through a

single receptor, ligands have been identified preferentially activate β-arrestin-mediated signaling

which is independent of G protein signaling (Rajagopal et al., 2010).

9

MATERIALS AND METHODS

Materials. Dulbecco's modified Eagle's media (DMEM) and Neurobasal media, B27

supplement, fetal bovine serum, and all other reagents used for cell cultures were purchased from

Invitrogen (Carlsbad, CA). All restriction enzymes used for molecular biology were purchased

from New England Biolabs (Ipswich, MA). Receptor agonists glutamate, aspartate, cysteic acid,

quisqualate, and DHPG, antagonists LY367385, 3-MATIDA, CPCCOEt, YM298198, JNJ

16259685, and inhibitors U73122, dynasore, U0126, and PD98059 were obtained from Tocris

Cookson (Ellisville, MO). Glutaric and succinic acids and all other chemicals were purchased

from Sigma (St. Louis, MO).

Cell cultures. CHO-K1 cells (American Type Culture Collection, Manassas, VA) were

stably transfected with mGlu1a receptor cDNA in pcDNA-3.1 vector (Invitrogen) using

Lipofectamine 2000 transfection reagent (Invitrogen). Cell lines were created from mass stable

transfections and selected with the antibiotic G-418 (Invitrogen). Cells were cultured in DMEM

supplemented with 10% fetal bovine serum, 2 mM glutamine, and 5% L-proline (Invitrogen).

Primary cultures of cerebellar granule neurons were prepared as described previously

(Wroblewski et al., 1985). Cerebella were dissected from 7-day old Sprague-Dawley rats and

granule cells were grown on poly-D-lysine coated 96-well plates (Nunc, Wiesbaden, Germany)

in Neurobasal medium containing B27 supplement, 2 mM glutamine, 100 g/ml gentamicin, and

either 5 mM KCl or 25 mM KCl. Cytosine arabinoside (10 µM) was added to granule cells the

day after plating to prevent growth of non-neural cells.

10

Transfection of cells with shRNA. SureSilencing shRNA plasmids against β-arrestin-1

(insert sequence: ATGGAGGAAGCTGATGATACT) and scrambled control were contained in

the pGeneClip Hygromycin vector (SABiosciences, Frederick, MD). CHO cells stably

expressing mGlu1a were transfected with plasmids containing shRNA using Effectene

transfection reagent (Quiagen, Valencia, CA) and selected in 0.8 mg/ml Hygromycin B.

Knockdown of β-arrestin-1 was confirmed by Western blotting. Rescue experiments were

performed by transfecting cells expressing shRNA with human β-arrestin-1, which was

subcloned in pcDNA-6.2 (Invitrogen).

Site directed mutagenesis. Introduction of point mutations in mGlu1a cDNA was made

using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Briefly, 20 ng

of plasmid containing mGlu1a cDNA was mixed with 125 ng of two mutagenic primers

(sequences in Table 3), dNTPs (50 μM) and 2.5 unit of Pfu DNA polymerase in a final volume

of 50 μl. Samples were denatured at 95°C for 30 sec and subjected to 20 cycles: denaturation

(95°C, 30 sec), annealing (55°C, 1 min), and elongation (72°C, 30 min), with a final 10 min

extension. Then 10 units of DpnI were added to digest the DNA template. After incubation at

37°C for 1 hour, samples were used for transformation of E. Coli XL1-Blue Supercompetent

cells. Positive clones were identified by restriction analysis and the authenticity of each mutation

was confirmed by DNA sequencing.

Western blots. Cells grown and treated in 35 mm dishes were collected in 25 mM Tris-

HCl buffer, pH 7.5 containing Halt protease and phosphatase inhibitor cocktails with 1 mM

EDTA (Pierce Biotechnology, Rockford, IL). Proteins were solubilized in Laemmli buffer

containing 50 mM DDT, and equal amounts of sample protein were resolved on 8%

11

polyacrylamide gels (Invitrogen). Proteins were transferred to Immobilon-P PVDF membranes

(Millipore, Billerica, MA) and were probed with antibodies against mGlu1a (BD Biosciences,

San Jose, CA), p44/42 MAPK (ERK1/2; Cell Signaling Technology, Danvers, MA), Phospho-

p44/42 MAPK (ERK1/2) (Thr202

/Tyr204

; Cell Signaling Technology), β-actin (Sigma), β-tubulin

(Sigma), and β-arrestin-1 (Abcam, Cambridge, MA). Proteins were visualized by incubation with

secondary antibodies coupled to horseradish peroxidase (Pierce) followed by exposure to

chemiluminescent HRP substrate SuperSignal West Femto (Pierce) and imaged with a CCD

camera. Blots were quantified using ImageJ software.

Receptor internalization assays. Receptor endocytosis was assayed as described

previously (Jeanneteau et al., 2006). Cells grown in 6 cm dishes were washed in PBS and labeled

for 30 minutes with cleavable Sulfo-NHS-SS-Biotin (Pierce), which was in a solution of PBS.

For 20 minutes, excess biotin was quenched with 100 mM glycine. Labeled cells were then

incubated for 30 minutes in the absence or presence of glutamate at 37oC. Stimulation was halted

by three washes with ice cold PBS. Extracellular biotin was then cleaved by 2 washes with 50

mM glutathione, 75 mM NaCl, 5 mM NaOH, 10% FBS in water for 15 minutes. Excess

glutathione was then quenched for 30 min in PBS containing 50 mM iodoacetamide and 1%

BSA. Cells were then lysed in RIPA buffer (0.5% Triton X-100, 10 mM Tris-HCl, pH 7.5, 120

mM NaCl, 25 mM KCl, and Halt protease inhibitor cocktails) and clarified, and biotinylated

proteins were precipitated with NeutrAvidin-agarose (Pierce). Proteins were rinsed 4 times with

RIPA buffer, solubilized in Laemmli buffer, and resolved by SDS-PAGE.

Measurement of ERK phosphorylation. Phosphorylated ERK was measured using cell-

based ELISA according to a protocol described previously (Versteeg et al., 2000). Cells were

12

grown and treated with agonists in 96 well plates. After incubation with agonist, cells were fixed

in 4% formaldehyde/PBS for 20 minutes at room temperature. After 3 washes in 0.1% Triton X-

100 (PBST) for membrane permeablization, endogenous peroxidase activity was quenched by 20

minutes incubation in PBS containing 0.5% H2O2 and 0.2% NaN3. After three more washes in

PBST, cells were blocked with 2% BSA for one hour and incubated overnight with primary

antibody against Phospho-p44/42 MAPK (ERK1/2) (Thr202

/Tyr204

) (Cell Signaling Technology).

Cells were then washed for five minutes three times in PBST and twice in PBS. A HRP-coupled

goat anti-rabbit secondary antibody (Pierce) was incubated for one hour at room temperature and

then cells were again washed five times. Cells were exposed to the colorimetric HRP substrate 1-

Step Ultra TMB (Pierce). After at least 10 minutes of developing, the reaction was stopped in 4

M H2SO4 and absorbance was read at 450 nm.

Assessment of cell viability. Viability of cells cultured on 96-well plates was measured by

incubation for 1 hour at 37oC with 0.2 mg/ml of 3-[4,5-dimethylthiazol-2-yl]-2,5-

diphenyltetrazolium bromide (MTT), which was purchased from Invitrogen. The formation of

the formazan product, proportional to the number of viable cells, was measured colorimetrically

at 570 nm after extraction with 70 µl DMSO (Mosmann, 1983).

Measurements of PI hydrolysis. Cells, cultured in 96-well plates, were incubated

overnight with 0.625 Ci/well myo-[3H]inositol (Perkin Elmer, Boston, MA) to label the cell

membrane phosphoinositides. After two washes with 0.1 ml of Locke’s buffer (156 mM NaCl,

5.6 mM KCl, 3.6 mM NaHCO3, 1 mM MgCl2, 1.3 mM CaCl2, 5.6 mM glucose and 20 mM

Hepes, pH 7.4), incubations with receptor ligands were carried out for 45 min at 37°C in Locke’s

buffer containing 20 mM LiCl to block inositol phosphate degradation. The reaction was

13

terminated by aspiration of media and inositol phosphates were extracted in 10 mM formic acid

for 30 min. Samples were transferred to opaque-welled plates and incubated with polylysine

coated yttrium scintillation proximity assay (SPA) beads (Perkin Elmer) and incubated at room

temperature for 1 hour with vigorous shaking. After 8 hours of incubation with SPA beads,

inositol phosphates were detected by scintillation counting.

Calculations and Statistics. For dose-response data, curves were fitted by nonlinear

regression to data points using the four-parameter logistic equation in Sigma Plot. Significance

testing was performed using Student’s t tests when comparing two groups or by Bonferroni-

corrected t tests when comparing multiple groups. Statistical significance was deemed by p <

0.01.

14

CHAPTER 2

The Protective Signaling of Metabotropic Glutamate Receptor 1 is Mediated by Sustained

β-Arrestin-1-Dependent ERK Phosphorylation

N.B. Many of the following results were previously published in the following manuscript:

Emery, A.C., Pshenichkin, S., Takoudjou, G.R., Grajkowska, E., Wolfe, B.B., Wroblewski, J.T.

(2010). The protective signaling of metabotropic glutamate receptor 1 is mediated by sustained,

beta-arrestin-1-dependent ERK phosphorylation. J Biol Chem, 285(34):26041-8.

15

INTRODUCTION

Metabotropic glutamate receptor 1 (mGlu1) is a G protein-coupled receptor that is linked to

various second messenger systems through its association with Gq. Numerous studies have

indicated a role of mGlu1 receptors in synaptic physiology, notably including systems of long-

term depression at the parallel fiber-Purkinje cell synapse in the cerebellum (Kano et al., 2008).

Additionally, several other studies indicate that mGlu1 receptors induce signaling that facilitates

cellular growth and development. In the presence of glutamate, mGlu1 receptors have been

shown to stimulate axon growth in the developing brain (Kreibich et al., 2004) and the outgrowth

of dendritic spines in the developing hippocampus (Vanderklish and Edelman, 2002).

Recent evidence has demonstrated that mGlu1 receptors stimulate signaling which protects

cultured cerebellar and cortical neurons from apoptosis (Pshenichkin et al., 2008). However, the

mechanisms of this neuroprotective signaling remained unknown. In these studies, the protective

effect of glutamate followed a different pharmacological profile than for PI hydrolysis, the

known signaling mechanism of the receptor. Therefore, the following studies were designed to

ascertain whether this signaling followed the known G protein-dependent signaling mechanism

or a novel G protein-independent mechanism.

The aim of these studies was to identify the signal transduction pathway through which

glutamate causes protective signaling in cells expressing mGlu1a receptors. The following data

determined that glutamate, but not quisqualate, stimulates a sustained phosphorylation of ERK

through mGlu1a receptors. This phenomenon was unique to mGlu1a receptors and required the

expression of β-arrestin-1. Moreover, inhibition of ERK phosphorylation and silencing of β-

16

arrestin-1 expression abolished the protective effects of glutamate. Therefore, the protective

signaling of mGlu1a receptors does not rely on the classical, G protein-mediated, mechanism of

signal transduction, but, instead involves a β-arrestin-dependent receptor internalization and

ERK phosphorylation.

17

RESULTS

Previous studies in primary cultures of cortical and cerebellar neurons have shown that

glutamate, acting selectively at mGlu1a receptors, rescued these neurons from apoptotic cell

death induced by conditions of trophic deprivation (Pshenichkin et al., 2008). This protective

effect of glutamate was revealed in the presence of antagonists of ionotropic glutamate receptors

which suppressed the excitotoxic actions of glutamate. Moreover, in these cells, the protective

effect was elicited by high concentrations of glutamate, about 10 times higher than those needed

to activate mGlu1-mediated PI hydrolysis.

In order to study the pharmacological and protective properties of mGlu1a without

interference from other glutamate receptors, CHO cells with stably expressed mGlu1a receptors

were used to assess the potency of mGlu1 agonists in producing the protective effect. Cells were

transferred to serum-free culture medium to induce apoptosis (Zhong et al., 1993) and their

viability was monitored by the MTT assay. Under these conditions, after 3 days, cells exhibited

approximately 35-50% viability relative to controls grown in serum-containing medium. Cells

exposed to 3 mM glutamate were fully protected from serum deprivation-induced apoptosis for

up to 6 days, having similar viability to control cells grown in serum-containing growth media

while lower concentrations of glutamate (0.3 mM) produced a partial protection from serum

deprivation (Figure 1). The protective effect of glutamate required the expression of mGlu1a

receptors since untransfected CHO cells were not protected from serum deprivation (Figure 2).

Also, among PLC-coupled mGlu receptors only mGlu1, but not mGlu5, was effective in

protecting from toxicity induced by serum deprivation (Figure 2).

18

Figure 1. Time course of protection by glutamate in CHO cells expressing mGlu1a

receptors. CHO cells stably expressing mGlu1a receptors were placed in serum-free media to

induce toxicity. In the presence of glutamate (Glu), these cells were protected from toxicity. Data

points represent means and error bars are S.E.M. from three independent time-course series of

MTT assays performed in triplicate.

Days Post-treatment

1 2 3 4 5 6

Via

bili

ty (

% o

f S

eru

m c

on

tro

l a

t d

ay 1

)

40

60

80

100

Serum

Serum-freeSerum-free + 0.3 mM Glu

Serum-free + 3 mM Glu

19

Figure 2. Efficacy of glutamate to protect CHO cells. CHO cells were stably transfected with

mGlu1a receptors or mGlu5a receptors cells and were subjected to serum deprivation for three

days in the presence or absence of 1 mM glutamate (Glu). Glutamate protected only CHO cells

expressing mGlu1a receptors. Values represent means and error bars represent S.E.M. from three

independent series of MTT assays performed in triplicate. **p<0.001 as compared to untreated

controls using Student's t-test.

CHO mGlu1a mGlu5a

Via

bili

ty (

% o

f S

eru

m c

on

tro

l)

0

20

40

60

80

100

**

Serum-free

Serum-free + 1 mM Glu

20

The addition of glutamate to the serum-free culture medium produced a dose-dependent increase

in viability with an EC50 of 153 + 31 μM; however, quisqualate, the most potent mGlu1 agonist,

was not effective in concentrations up to 3 mM (Figure 3). In order to further ascertain that the

protective effect is in fact due to the activation of mGlu1a receptors, and not to a nonspecific

effect involving glutamate transporters or another mechanism, glutamate-induced protection was

tested in the presence of three distinct noncompetitive mGlu1-selective antagonists. Application

of 100 μM CPCCOEt, 10 μM YM-298198, or 30 μM JNJ16259685 completely inhibited the

activity of 1 mM glutamate (Figure 4). Effective concentrations of these antagonists were

derived from pilot experiments evaluating their effects on mGlu1 receptor-mediated PI

hydrolysis (data not shown). A more detailed analysis showed that YM-298198 inhibited

glutamate-induced protection in a noncompetitive manner with increasing concentrations of the

antagonist decreasing glutamate efficacy but having no effect on its potency as shown by similar

glutamate EC50 values in the presence of different concentrations of antagonist (Figure 5, Table

1).

The observed pharmacological properties of mGlu1a in inducing the protective effect were

inconsistent with its known properties in stimulating G protein-mediated phosphoinositide (PI)

hydrolysis (Aramori and Nakanishi, 1992). To study this discrepancy, the potency of the agonists

to stimulate PI hydrolysis in CHO cells expressing mGlu1a that were used in these experiments

was determined. As shown in Figure 6, both glutamate and quisqualate stimulated PI hydrolysis

in a dose-dependent manner with quisqualate being more potent (EC50 = 0.63 ± 0.13 μM) than

glutamate (EC50 = 16 ± 1.2 μM). Hence, the potency of glutamate to induce protection was

approximately 10 times lower than its potency to stimulate PI hydrolysis.

21

[Agonist], M

10-5 10-4 10-3 10-2

Via

bility (

% o

f u

ntr

eate

d c

ontr

ol)

0

20

40

60

80 Glu

Quis

Figure 3. Potency of glutamate to protect CHO cells expressing mGlu1a receptors. CHO

cells stably expressing mGlu1a receptors were subjected to serum withdrawal for three days in

the absence or presence of receptor agonists. Glutamate (Glu) was effective, EC50 = 154 ± 31

μM, whereas quisqualate (Quis) failed to protect cells at concentrations as high as 3 mM. Data

points are means and error bars are S.E.M. from three independent MTT assays performed in

triplicate.

22

Figure 4. Inhibition of the protective effect of glutamate by mGlu1-selective noncompetitive

antagonists. CHO cells stably expressing mGlu1a receptors were subjected to serum withdrawal

for three days in the absence or presence of 1 mM glutamate (Glu). CP –CPCCOEt (100 μM),

YM – YM-298198 (10 μM), and JN – JNJ16259685 (30 μM). Values are means from at least

three independent experiments performed in triplicate with error bars representing S.E.M.

**p<0.001 as compared to untreated controls using Student's t-test.

Control CP YM JNJ

Via

bili

ty (

% o

f se

rum

co

ntr

ol)

0

50

100

Serum-free

Serum-free+Glu

**

23

Figure 5. Noncompetitive inhibition of protection by glutamate. Dose-response curves of

glutamate-mediated protection in the presence of varying concentrations of YM-298198 (YM).

Cell viability was measured by MTT assay after 72 hours of incubation with the ligands in

serum-free conditions and is expressed as a percent of cells viable in parallel cultures grown in

the presence of serum. All data points are means from at least three independent experiments

performed in triplicate with error bars representing S.E.M.

[Glutamate], M

10-4 10-3 10-2

Via

bili

ty (

% o

f u

ntr

ea

ted

con

trol)

40

60

80

100YM = 0

YM = 0.03 M

YM = 0.3 M

YM = 3 M

24

Protection from toxicity PI hydrolysis

YM-298198 EC50 (μM) Emax (% Control) EC50 (μM) Emax (% Basal)

(μM) Avg ± SEM Avg ± SEM Avg ± SEM Avg ± SEM

0 191 ± 27 93 ± 1.7 28 ± 3.1

259 ± 5.0

0.03 180 ± 11 74 ± 3.6* 42 ± 4.8* 233 ± 16

0.3 160 ± 56 63 ± 2.4* 94 ± 6.0* 198 ± 14*

3 153 ± 20 53 ± 0.9* 171 ± 11* 157 ± 12*

Table 1. Summary of mGlu1a receptor pharmacology. Comparison of potency and efficacy of

glutamate to induce protection and stimulate PI hydrolysis in the absence and presence of a non-

competitive antagonist YM-289198 in CHO cells expressing mGlu1a receptors. Potency (EC50)

refers to the concentration of glutamate producing a half-maximal effect. Efficacy (Emax)

represents the maximal effect of glutamate expressed as a percent of control cell viability in the

presence of serum for toxicity experiments and as a percent of basal activity for measurements of

PI hydrolysis. Data are averages ± SEM calculated from dose-response curves obtained in 3-6

experiments. *p<0.05 compared to values obtained in the absence of antagonist using

Bonferroni-adjusted t-test.

25

Figure 6. Pharmacology of PI hydrolysis through mGlu1a receptors. In CHO cells stably

expressing mGlu1a receptors, PI hydrolysis is activated in a dose-dependent manner by

glutamate (Glu; EC50 = 11 ± 1.2 μM) and quisqualate (Quis; EC50 = 0.63 ± 0.13 μM). Glutamate-

induced PI hydrolysis is noncompetitively inhibited by 10 M YM298198 (YM). Data points

represent means from three independent experiments performed in triplicate with error bars

corresponding to S.E.M.

[Agonist], M

10-8 10-7 10-6 10-5 10-4 10-3 10-2

PI

hyd

roly

sis

(%

of

ba

sa

l)

100

200

300

400

Glu

Quis

Glu + YM

26

One possible explanation for these differences would be that the potencies observed in

protection reflect an artefactual decrease of agonist concentrations occurring during the 3 day

incubation that would not appear in the 45 minute incubation used when measuring PI

hydrolysis. In control experiments testing agonist stability, glutamate and quisqualate were

incubated with CHO cells for 3 days and then used to stimulate PI hydrolysis. As shown in

Figure 7, both conditioned and freshly prepared agonists stimulated PI hydrolysis with the same

potency, demonstrating that these compounds remain intact for the entire 3 day incubation and

that the observed discrepancies do not result from a decrease in agonist concentration.

As expected, glutamate-stimulated PI hydrolysis was abolished by the selective

noncompetitive mGlu1 antagonist YM-298198 and the antagonism appeared noncompetitive as

seen by the decrease of the maximal effect of glutamate with increasing concentrations of the

antagonist (Figure 8). However, in contrast with the data obtained for glutamate protective

signaling, the calculated potency of glutamate in PI hydrolysis experiments decreased with

increasing antagonist concentrations (Table 1). Usually, such right-shifts of agonist potency in

the presence of a noncompetitive antagonist indicate the existence of receptor reserve (also

known as spare receptors) and are frequently seen in heterologous expression systems. This leads

to the conclusion that the apparent difference in potency of glutamate to produce both effects

results from the presence of a high receptor reserve for PI hydrolysis but no receptor reserve for

protective signaling. The use of the highest concentration of YM-298198 in PI hydrolysis

experiments increases the EC50 of glutamate to the levels observed in protection experiments.

Hence, once this receptor reserve is abolished by the use of a noncompetitive antagonist the

potency of glutamate to produce both effects is the same.

27

Figure 7. Bioassay demonstrating the stability of glutamate and quisqualate. Stimulation of

PI hydrolysis in CHO cells expressing mGlu1a receptors by fresh agonists and agonists

conditioned in the presence of CHO cells for 3 days. EC50 for fresh and conditioned agonists was

as follows: glutamate (19 ± 3.2 μM and 12 ± 2.6 μM) and quisqualate (0.23 ± 0.11 μM and 0.24

± 0.13 μM). All data points are means from three independent experiments performed in

triplicate with error bars representing S.E.M.

28

[Glutamate], M

10-6 10-5 10-4 10-3

PI h

yd

roly

sis

(%

of b

asa

l)

100

150

200

250YM = 0

YM = 3 nM

YM = 30 nM

YM = 300 nM

Figure 8. Receptor reserve for mGlu1a receptor-mediated PI hydrolysis. Dose-response

curves of glutamate-induced PI hydrolysis in the presence of varying concentrations of mGlu1-

selective noncompetitive antagonist YM-298198 (YM). With increasing concentrations of YM,

the EC50 for glutamate increases (see Table 1), indicating the presence of receptor reserve. All

data points are means from three independent experiments performed in triplicate with error bars

representing S.E.M.

29

While these pharmacological results confirm that the protective signaling and PI hydrolysis are

mediated by the same receptor, they also indicate the existence of ligand bias in producing the

two effects. Ligand bias is defined as the ability of some agonists, such as quisqualate, to activate

only selected signal transduction mechanisms while other agonists, such as glutamate, may

activate all signaling associated with this receptor (Kenakin, 2007).

Signal transduction of protective signaling through mGlu1.

To identify the mechanism through which mGlu1a elicits protective signaling, a selective

inhibitor of signaling which occurs downstream of G protein activation was used. Since mGlu1

typically couples to phospholipase C (PLC) through Gq/11 (Aramori and Nakanishi, 1992),

transfected CHO cells were treated with the PLC inhibitor U73122 (Bleasdale et al., 1990). In

conditions of serum deprivation for 3 days U73122 (30 μM) failed to block the protective effect

of glutamate (Figure 9A). In control experiments, 30 μM culture-conditioned U73122 effectively

blocked glutamate-induced PI hydrolysis (Figure 9B), indicating that this compound is stable in

these culture conditions and is effective in blocking PI hydrolysis in this model. These data,

therefore, indicate that the protective effect is not mediated by the coupling of mGlu1a receptors

to PLC. These data suggest that the protective effect mediated by mGlu1a receptor is not related

to its ability to stimulate PI hydrolysis but, rather, is mediated by a different signal transduction

mechanism.

30

A B

Figure 9. Glutamate-induced protection is PLC-independent but MEK and dynamin-

dependent. (A) CHO cells expressing mGlu1a were subjected to serum deprivation for 72 hours

in the presence of glutamate (Glu) and inhibitors U73122 (30 μM), PD98059 (10 μM), U0126

(10 μM), or dynasore (100 μM). Phospholipase C inhibitor U73122 fails to block protection by

glutamate, whereas protection is blocked by MEK1 inhibitor PD98059, MEK 1/2 inhibitor

U0126, and dynamin inhibitor, dynasore. ** p < 0.001 as compared with serum-deprived

controls using Bonferroni-adjusted t test. (B) Inhibition of glutamate-induced PI hydrolysis in

mGlu1a-expressing CHO cells by U73122 (30 μM) that is freshly prepared (U73) or

preconditioned (U73-C) by exposure to CHO cells for 3 days, indicating U73122 is stable for the

entire 3 day incubation period. Values are means from at least three independent experiments

performed in triplicate with error bars representing S.E.M.

Control U73122 PD98059 U0126 Dynasore

Via

bili

ty (

% o

f S

eru

m c

on

tro

l)

0

20

40

60

80

100

Serum Serum-free + 0.3 mM Glu + 3 mM Glu

****

** **

PI

hyd

roly

sis

(%

of

ba

sa

l)

0

100

200

300

Con U73 U73-C

31

Since mGlu1 has also been reported to activate the MAP kinase pathway (Sheffler and Conn,

2008), the involvement of this pathway in the protective effect of glutamate was investigated.

Indeed, treatment of mGlu1a-expressing CHO cells with MEK1 inhibitor PD98059 (Rosen et al.,

1994) or with the MEK1/2 inhibitor U0126 (Favata et al., 1998) abolished the efficacy of

glutamate to cause protective signaling (Figure 9A).

Furthermore dynasore, an inhibitor of dynamin, a protein in the endocytotic pathway for

mGlu1 (Mundell et al., 2001), also abolished glutamate-induced protection. In control

experiments, monitoring receptor trafficking, dynasore (100 μM) blocked glutamate-induced

internalization of mGlu1 receptors (Figure 10A, 10B). These data suggest that the protective

effect of glutamate is not mediated by the classical G protein-mediated mechanism, but instead, a

G protein-independent activation of MAP kinase pathway, most probably involving receptor

internalization and the phosphorylation of ERK. Additionally, these studies were replicated in

cultured cerebellar granule cells, which natively express mGlu1 receptors (Pshenichkin et al.,

2008). Cerebellar granule cells require 25 mM extracellular potassium (K25) to survive in

culture. Reducing the potassium concentration to 5 mM (K5) caused granule cells to exhibit an

approximately 50% reduction in viability compared to K25 controls (Figure 11). Glutamate

protected these cells from low potassium-induced apoptosis, and protection was inhibited by

both MEK inhibitors, 30 M U0126 and 30 M PD98059, as well as dynasore (100 M). As

with transfected CHO cells, the PLC inhibitor U73122 (30 M) failed to attenuate the protective

effect of glutamate (Figure 11).

32

A B . Control Dynasore 100 μM

Glu (1 mM) + + - - + + - -

Fraction P S P S P S P S

Figure 10. Internalization of mGlu1a receptors is dynamin-dependent. (A) Inhibition of

glutamate-induced internalization of mGlu1a receptors by the dynamin inhibitor dynasore (100

μM). Cells were labeled with cleavable biotin and stimulated with 1 mM glutamate for 1 hour.

Extracellular biotin was removed and internalized mGlu1a receptors were pulled down in

NeutrAvidin. P, pellet (internalized during incubation); S, supernatant (remaining receptors). (B)

Densitometric quantification of Western blot from Fig. 10A.

% o

f to

tal m

Glu

1 a

mou

nt

0

20

40

60

80

100

% Internalized

% Remaining

Glu GluBasal Basal

Control Dynasore (100 M)

33

Control U73122 PD98059 U0126 Dynasore

Via

bili

ty (

% o

f K

25 C

ontr

ol)

0

20

40

60

80

100

K25

K5

0.1 mM Glu

1 mM Glu

Figure 11. Glutamate-induced protection is PLC-independent but MEK and Dynamin-

dependent in neurons. Cerebellar granule cells were subjected to low potassium (K5) for 12

hours to induce apoptosis in the presence of glutamate (Glu) and inhibitors U73122 (30 μM),

PD98059 (30 μM), U0126 (30 μM), or dynasore (100 μM). Phospholipase C inhibitor U73122

fails to block protection by glutamate, whereas protection is blocked by MEK1 inhibitor

PD98059, MEK 1/2 inhibitor U0126, and dynamin inhibitor, dynasore.

34

Time-course and pharmacology of mGlu1- mediated ERK phosphorylation.

Previous reports have demonstrated that the stimulation of group I mGlu receptors causes

transient ERK phosphorylation that occurs due to G protein activation and subsides within 30

minutes of agonist application (Karim et al., 2001). As shown by Western blot (Fig. 12A, 12B),

in CHO cells expressing mGlu1, glutamate and quisqualate increased ERK phosphorylation at 5

minutes after agonist application. In addition, when agonists were added for 24 hours, glutamate,

but not quisqualate, produced a sustained ERK phosphorylation. As shown in Fig. 12A, both

isoforms of ERK were equally phosphorylated.

These effects were further quantified using an ELISA-based assay to compare the levels of

ERK phosphorylation in CHO cells expressing either mGlu1a or mGlu5a receptors. In cells

expressing mGlu5a both glutamate and quisqualate elicited a transient ERK phosphorylation

which was elevated at 5 minutes after the addition of agonists, and subsided after 30 minutes

(Figure 13B). However, in cells expressing mGlu1a, while both agonists induced a transient

increase in ERK phosphorylation, after the addition of glutamate ERK phosphorylation was

elevated up to 24 hours (Figure 13A).

A closer look at the pharmacology of ERK phosphorylation revealed that the transient, 5 minute,

phosphorylation was induced by both glutamate and quisqualate (Figure 14A), with a

pharmacological profile and agonist potencies similar to those observed for PI hydrolysis (Figure

6). Also like PI hydrolysis, agonist-induced ERK phosphorylation was abolished by the mGlu1-

selective noncompetitive antagonist YM-298198 (Figure 14A). In contrast, the sustained 24 hour

ERK phosphorylation was only induced by glutamate, but not by quisqualate, and was blocked

by YM-298198 (Figure 14B).

35

A B

Figure 12. ERK phosphorylation due to mGlu1 agonists. (A) Representative Western blot of

phosphorylated ERK (pERK) and total ERK (ERK) at 5 minutes and 24 hours after the

application of glutamate or quisqualate to CHO cells expressing mGlu1a receptors. (B)

Densitometric quantification of Western blot from A, expressed as the ratio of measured

intensities for the sum of pERK bands divided by total ERK. 12-O-tetradecanoylphorbol-13-

acetate (TPA), which activates PKC, was used as positive control.

24 h 5 min 24 h 5 min 24 h 5 min 24 h 5 min

Ra

tio

pE

rk/t

ota

l E

rk

0

1

2

3

Glu Quis TPABasal

36

A mGlu1 B mGlu5

Figure 13. ELISA-based quantification of phosphorylated ERK. CHO cells expressing

mGlu1a receptors (A) or mGlu5a receptors (B) were treated with agonists glutamate (Glu) or

quisqualate (Quis) for varying times. Values are means from at least three experiments

performed in triplicate with error bars representing S.E.M. and are expressed as the percent of

ERK phosphorylation measured in cells not treated with agonists. *, p < 0.01 and **, p < 0.001

as compared with untreated controls using Bonferroni-adjusted t test.

Time

5 min 30 min 24 h

ph

osp

ho

ER

K (

% o

f b

asa

l)

100

150

200

250

Glu 1 mM

Glu 10 mM

Quis 1 M

**** **

**

**

**

**

*

Time

5 min 30 min 24 h

phosphoE

RK

(%

of

basal)

100

150

200

250Glu 1 mM

Glu 10 mM

Quis 1 M

**

** **

*

37

A 5 minutes B 24 hours

Figure 14. Agonist profiles of ERK phosphorylation through mGlu1a receptors. CHO cells

expressing mGlu1a receptors were stimulated with agonists glutamate (Glu) or quisqualate

(Quis) for 5 minutes (A) or 24 hours (B). Glutamate caused an increase in phosphorylated ERK

at both times, while quisqualate was only active at the 5 minute time point. All effects were

inhibited by 10 M YM298198 (YM). All data points are means from at least three experiments

performed in triplicate with error bars representing S.E.M. and are expressed as the percent of

ERK phosphorylation measured in cells not treated with agonists.

[Agonist], M

10-7 10-6 10-5 10-4 10-3 10-2

Ph

osp

ho

ER

K (

% o

f b

asa

l)

100

200

300

Glu

Quis

Glu+YM

Quis+YM

[Agonist], M

10-5 10-4 10-3 10-2

Pho

sph

oE

RK

(%

of

ba

sal)

100

150

200

250Glu

Quis

Glu+YM

38

The observed agonist potencies were lower than those for PI hydrolysis and transient ERK

phosphorylation, but approximated agonist potencies observed for the protective signaling

through mGlu1a (Figure 3).

Together, these data indicate that transient (5 minute) ERK phosphorylation is activated by

both mGlu1a and mGlu5a receptors and has a pharmacological profile similar to the activation of

PI hydrolysis, hence is mediated by a G protein-mediated mechanism. In contrast, the sustained

ERK phosphorylation, lasting up to 24 hours, is only activated by mGlu1a, and only glutamate is

able to produce the protective mGlu1a signaling. These results further support the existence of a

ligand bias which extends to ERK phosphorylation and reveals the existence of two distinct

signal transduction mechanisms. The classical mechanism, activated by both glutamate and

quisqualate, mGlu1 agonists with an apparent high potency, involves a G protein-mediated PI

hydrolysis and transient ERK phosphorylation. The second mechanism, which correlates well

with protective mGlu1a signaling, is activated with an apparent lower potency by glutamate and

is mediated by a G protein-independent mechanism which leads to prolonged activation of

MAPK pathway and sustained ERK phosphorylation.

39

The role of β-arrestin-1 in sustained ERK phosphorylation and the protective signaling of

mGlu1a.

Based on similarities with other GPCRs (Brauner-Osborne et al., 2007) we hypothesized that

the mGlu1a-induced phosphorylation of ERK may involve a β-arrestin-1-dependent

internalization of mGlu1a receptors and formation of a signaling complex mediating this

protective signaling. To investigate this hypothesis the expression of β-arrestin-1 in mGlu1a-

expressing CHO cells was silenced using a plasmid encoding shRNA targeted against rat β-

arrestin-1. Transfection with this plasmid encoding shRNA caused a substantial decrease in

protein expression levels of β-arrestin-1 as monitored by Western blots, while a scrambled

shRNA used in control experiments had no effect (Figure 15A, 15B). In rescue experiments,

cells expressing targeted shRNA were transfected with cDNA for human β-arrestin-1, which is

refractory to the silencing effects of rat shRNA due to having mismatches of several bases.

Expression of human β-arrestin-1 caused an approximate 3-fold increase of β-arrestin-1 protein

levels relative to untransfected controls (Figure 15A, 15B). Additional control experiments

showed that shRNA silencing of β-arrestin-1 failed to decrease agonist-induced PI hydrolysis in

CHO cells expressing mGlu1a receptors (Figure 16), suggesting that this shRNA did not

significantly affect classical mGlu1 receptor function. In contrast, β-arrestin-1 shRNA effectively

abolished the protective effect of glutamate against serum deprivation (Figure 16). Protection by

glutamate was restored after transfection of human β-arrestin-1 (Figure 17).

40

Figure 15. Silencing of -arrestin-1 with shRNA. (A) Protein levels of β-arrestin-1 in mGlu1a-

expressing CHO cells. Samples in lane 3 were transfected with plasmids encoding shRNA

targeted against rat β-arrestin-1, which reduces expression to 24% of untransfected controls (lane

1). Scrambled control shRNA does not affect protein levels of β-arrestin-1 (lane 2). Knockdown

due to shRNA is reversed by transfection of human β-arrestin-1 (lane 4). Knockdown due to

shRNA is reversed by transfection of human β-arrestin-1 (lane 4). Transfection with human β-

arrestin-1 causes expression levels of 314% relative to untransfected controls. (B) Densitometric

quantification of the Western blot expressed as a ratio of band densities measured for β-arrestin-1

and β-actin.

41

Figure 16. Knockdown of β-arrestin-1 of does not impair classical mGlu1a receptor

signaling. PI hydrolysis was measured in CHO cells expressing mGlu1a and scrambled shRNA;

or mGlu1a and shRNA targeted against β-arrestin-1, which were stimulated with agonists

glutamate (Glu) or quisqualate (Quis). All data points are means from two experiments

performed in quadruplicate with error bars representing S.E.M.

2D Graph 1

[Agonist], M

10-8 10-7 10-6 10-5 10-4 10-3

PI

hydro

lysis

(%

of

basal)

100

200

300

400

Glu

Quis

shRNA+Glu

shRNA+Quis

42

Figure 17. Protective signaling through mGlu1a is abolished by shRNA silencing of β-

arrestin-1. In shRNA-expressing cells, transfection of human β-arrestin-1 restores protective

signaling by glutamate. All CHO cells expressed mGlu1a receptors and were treated with serum

deprivation for three days to induce toxicity, which was measured by MTT assays. Data points

reflect experiments performed at least four times in triplicate with data points representing means

and error bars as S.E.M.

9110

[Glutamate], M

10-6 10-5 10-4 10-3 10-2

Via

bility (

% o

f S

eru

m)

40

60

80

100

Scrambled shRNA

shRNA

shRNA +Ar-1

43

Internalization assays showed that a 30 minute pulse of 1 mM glutamate caused 50% of

mGlu1a receptors to become internalized in contrast to control cells, where approximately 18%

of mGlu1a receptors were internalized during the 30 minute control incubation (Figure 17A,

17B). Transfection with shRNA targeted toward β-arrestin-1 caused internalization in the

presence of 1 mM glutamate to be equivalent with the constitutive internalization seen in cells

not exposed to glutamate (Figure 18A, 18B).

Further experiments showed that β-arrestin-1 silencing differentially affected the transient and

sustained ERK phosphorylation mediated by mGlu1a receptors. The transient ERK

phosphorylation induced by glutamate and quisqualate was reduced by the PLC inhibitor

U73122 but not by β-arrestin-1 silencing (Figure 19A). In contrast, the sustained ERK

phosphorylation, measured 24 hours after the application of agonist, was activated only by

glutamate and was abolished by β-arrestin-1 silencing, but not by the PLC inhibitor U73122

(Figure 19B).

These results show that the classical mGlu1a receptor signaling through PI hydrolysis, which

also includes transient ERK phosphorylation, does not depend on the presence of β-arrestin-1. In

fact, Figure 16 suggests that PI hydrolysis after silencing of β-arrestin-1 was slightly elevated,

which corresponds well to the reported ability of β-arrestin to inhibit G protein-mediated signal

transduction (Diviani et al., 1996). In contrast, these results indicate that the presence of β-

arrestin-1 is required for both the sustained ERK phosphorylation and the protective mGlu1a

signaling.

44

A. B.

Figure 18. Endocytosis of mGlu1a receptors is β-arrestin-1-dependent. (A) Representative

Western blot showing that glutamate (Glu)-induced internalization of mGlu1a is attenuated by

shRNA silencing of β-arrestin-1. CHO cells expressing mGlu1a receptors were labeled with

cleavable biotin and stimulated with 1 mM glutamate for 1 hour. Extracellular biotin was

removed and internalized mGlu1a receptors were pulled down in NeutrAvidin. P, pellet

(internalized during incubation); S, supernatant (remaining receptors). (B) Densitometric

quantification of three independent internalization assays.

% o

f to

tal m

Glu

1 a

moun

t

0

20

40

60

80

100

% Internalized

% Remaining

Glu GluBasal Basal

Scrambled shRNA Arrestin-1 shRNA

45

A. B.

Figure 19. Sustained ERK phosphorylation through mGlu1a receptors is β-arrestin-1-

dependent. Effect of shRNA silencing of β-arrestin1 on ERK phosphorylation induced by

agonists glutamate (Glu) and quisqualate (Quis) after 5 minutes (A) and 24 hours (B). Transient

ERK phosphorylation is sensitive to the inhibition of PLC by U73122 (30 μM) (A). Sustained

ERK phosphorylation is blocked by shRNA silencing of β-arrestin-1 but is not affected by the

inhibition of PLC (B). ERK phosphorylation was measured using ELISA, and the data are

expressed as a percent of ERK phosphorylated in the absence of agonists. All values are means

from at least three experiments performed in triplicate with error bars representing S.E.M. **p <

0.001) as compared with untreated controls using Bonferroni-adjusted t test.

46

Figure 20. Schematic representation of signal transduction through mGlu1 receptors.

During short-term treatment with glutamate, mGlu1 receptors stimulate PI hydrolysis via

coupling to Gq. After long-term treatment, the receptor is hypothesized to be phosphorylated by

a GRK and is internalized in by dynamin and -arrestin-1 (arr-1). Once internalized, mGlu1

receptors cause a long-term phosphorylation (P) of MEK and ERK, which leads to cellular

survival.

47

DISCUSSION

Previously it has been shown that glutamate, acting selectively at mGlu1 receptors, protects

neurons from apoptotic death induced by trophic deprivation (Pshenichkin et al., 2008). This

study demonstrates the existence of a novel signal transduction mechanism that allows mGlu1

receptors to mediate this protective signaling. This mechanism is distinct from the well described

classical G protein-mediated signal transduction of group I mGlu receptors (Aramori and

Nakanishi, 1992; Pickering et al., 1993) and is not shared by mGlu5 receptors, likely due to

differences in the two receptors respective C-termini. Shown here is that in CHO cells expressing

mGlu1a receptors, glutamate caused protection from toxicity due to serum deprivation, and this

protection was blocked not only by noncompetitive antagonists of mGlu1, but also by the

inhibition of receptor internalization by silencing β-arrestin-1 expression and inhibition of

dynamin. Furthermore, protective signaling was blocked by inhibitors of MEK1/2. The pathway

through which mGlu1 acts appears to be unrelated to classical G protein signaling, as protection

was not attenuated by inhibition of phospholipase C. Instead, these data reveal the existence of a

previously unreported signaling mechanism associated with mGlu1a receptors, namely a G

protein-independent protective signaling pathway that requires β-arrestin-1 and activation of

MEK, followed by ERK phosphorylation.

Anti-apoptotic signaling through β-arrestin has been demonstrated for numerous GPCRs

(DeWire et al., 2007). As signaling molecules, β-arrestins have been shown to mediate the

stimulation of phosphorylation of several protein kinases, including ERK (Daaka et al., 1998).

Additionally, β-arrestin-mediated signaling has been shown to be independent of G protein

signaling for multiple receptors including the angiotensin II type 1a receptor (Luttrell et al.,

48

2001), the D3 dopamine receptor (Beom et al., 2004), the β2-adrenergic receptor (Shenoy et al.,

2006), and the mu opioid receptor (Macey et al., 2006). Consistent with previous reports that β-

arrestin-1 is required for agonist-induced internalization of mGlu1 (Dale et al., 2001), these

findings suggest that the protective signaling of mGlu1a may be initiated by agonist-dependent

receptor internalization, followed by a non-classical, G protein-independent mechanism of signal

transduction. The current results also indicate that, similarly to several other GPCRs (Kenakin,

2007), mGlu1a receptors show a ligand bias towards the different signaling mechanisms,

manifested by the ability of glutamate to activate both signaling pathways, while quisqualate

activates only G protein-mediated PI hydrolysis.

The protective mGlu1a signaling appears to be mediated by a different mechanism of ERK

phosphorylation than that described previously. Several reports show that agonists acting at

mGlu1 receptors induce a transient increase of ERK phosphorylation which peaks at 5 minutes

and subsides within 30 minutes (Mundell et al., 2001). Consistent with these reports, in CHO

cells expressing mGlu1, at 5 minutes, agonists induced a transient ERK phosphorylation which

had a pharmacological profile similar to that observed for PI hydrolysis, was blocked by the

inhibition of PLC activity, and was not dependent on β-arrestin expression. At higher glutamate

concentrations, β-arrestin silencing slightly reduced ERK phosphorylation also at 5 minutes,

which is consistent with a previous report showing that a portion of mGlu1-mediated transient

MAP kinase phosphorylation is β-arrestin-dependent (Iacovelli et al., 2003). Together, these data

suggest that transient ERK phosphorylation is mediated by the classical mechanism of G protein-

mediated PI hydrolysis.

49

In addition, these data show that glutamate, acting at mGlu1 receptors, activates also a long

lasting phase of ERK phosphorylation, which was maintained in the presence of glutamate for 24

hours. This is the first report of a sustained phosphorylation of ERK due to stimulation of a

metabotropic glutamate receptor. Similar to the protective effect of glutamate, the sustained

phase of ERK phosphorylation was insensitive to PLC inhibition and was completely abolished

by shRNA silencing of β-arrestin-1 expression. In contrast to transient ERK phosphorylation, the

sustained phase was induced by much higher glutamate concentrations and was not elicited by

quisqualate. As demonstrated in these studies, this difference in glutamate potency reflected the

presence in transfected CHO cells of receptor reserve for PI hydrolysis. The pharmacological

profile of the sustained ERK phosphorylation resembled that of the protective mGlu1a signaling,

but not that of PI hydrolysis and transient ERK phosphorylation. These data strongly suggest that

the protective mGlu1a signaling is mediated by glutamate-induced sustained ERK

phosphorylation. While the specific downstream targets of phosphorylated ERK have not been

identified, phosphorylated ERK, acting at the cell nucleus, was shown to cause protective

cellular responses (Seger and Krebs, 1995).

50

CHAPTER 3

Metabotropic Glutamate Receptor 1 Possesses Two Glutamate Binding Sites Coupled to

Different Signal Transduction Systems

51

INTRODUCTION

The previous chapter demonstrated that in CHO cells transfected with mGlu1a receptors,

stimulation of mGlu1a receptors with glutamate protected cells from serum withdrawal-induced

apoptosis. These results were published in Emery et al., 2010. In this manuscript we showed that,

protective signaling through mGlu1a receptors was accomplished by a β-arrestin-1-dependent

sustained phosphorylation of ERK. Moreover, this study on signal transduction of mGlu1a

receptors indicates that classical, G protein-mediated signal transduction and transient ERK

phosphorylation differ in their pharmacological profiles from sustained ERK phosphorylation

and protective signaling. In these studies, the classical mGlu1a-mediated PI hydrolysis and

transient ERK phosphorylation was induced by both quisqualate and glutamate with quisqualate

as the most potent agonist.

Our data are consistent with several previous reports on the pharmacology of mGlu1-

mediated PI hydrolysis (Aramori and Nakanishi, 1992) and ERK phosphorylation (Thandi et al.,

2002). In contrast, sustained ERK phosphorylation and protection followed a unique

pharmacological profile. Only glutamate produced these effects, demonstrating an apparent

ligand bias at mGlu1a receptors. Ligand bias is a new and emerging concept in pharmacology

that has been shown to exist for other GPCRs when various agonists preferentially activate

receptor conformations that are selectively conducive for different signal transduction pathways

(Rajagopal et al., 2010). The earliest reported example of ligand bias was in M1 muscarinic

receptors, which activate both cAMP and PLC in response to their endogenous agonist

acetylcholine. However, several ligands were identified that selectively activate PLC while

blocking cAMP formation through these receptors (Fisher et al., 1993). Ligand bias, also referred

52

to as “biased agonism”, has since been reported in numerous GPCRs, and has been recently

reviewed by Rajagopal et al. (2010). In addition to selective activation of different G protein-

dependent pathways, ligands have been identified which preferentially activate β-arrestin-

mediated signaling which is independent of G protein signaling for several receptor systems

(Wei et al., 2003).

Our previous results demonstrate that sustained ERK phosphorylation and protective

signaling are β-arrestin-1-dependent, and this may suggest that agonists that do not cause these

effects, such as quisqualate, are biased toward PI hydrolysis through mGlu1 receptors, whereas

an agonist such as glutamate is less biased owing to the observations that glutamate activated

both signal transduction pathways. While the structural properties responsible for the observed

ligand bias are unknown, the X-ray crystallographic studies of the ligand binding domain of

mGlu1 (Kunishima et al., 2000), have led to the suggestions that glutamate, the apparently

unbiased ligand, interacts with 14 amino acid residues while quisqualate, the apparently biased

ligand, interacts with only nine of these residues (Sato et al., 2003). The interaction of glutamate

with additional residues in the binding domain may represent a mechanism by which glutamate,

but not quisqualate, can activate the two separate signal transduction pathways. This raises an

interesting possibility that it may be possible to identify compounds which activate mGlu1

receptors but are biased toward sustained ERK phosphorylation and cellular survival. The aim of

the following studies is to identify the mechanism of ligand bias by which glutamate, but not

quisqualate causes protective signaling in cells expressing mGlu1a receptors. In these studies,

glutaric and succinic acids are identified as compounds which act as ligands at mGlu1a receptors

that are completely biased toward sustained ERK phosphorylation and protective signaling and

53

are inactive in stimulating PI hydrolysis. These dicarboxylic acids may serve as lead compounds

for drug discovery.

54

RESULTS

Previous studies in primary cultures of cortical neurons and cerebellar granule neurons

have demonstrated that glutamate, acting selectively at mGlu1 receptors, enhanced PI hydrolysis,

but also rescued these neurons from apoptotic cell death induced by conditions of trophic

deprivation (Pshenichkin et al., 2008). This protective effect of glutamate was revealed in the

presence of antagonists of ionotropic glutamate receptors which suppressed the excitotoxic

actions of glutamate. Studies in CHO cells stably transfected with mGlu1a receptors have shown

that protective signaling of mGlu1a is not mediated by its coupling with G proteins, but instead,

it is due to β-arrestin-1-mediated sustained ERK phosphorylation (Emery et al., 2010). This

sustained ERK phosphorylation is distinct from the transient, G protein–mediated and PLC-

dependent phosphorylation of ERK also seen after activation of mGlu1a receptors. In both

cerebellar granule cells, which express native mGlu1a receptors, and in CHO cells transfected

with mGlu1a receptors, protective signaling through mGlu1a receptors was induced only by

glutamate, the native ligand for the receptor, but not by quisqualate (Emery et al., 2010;

Pshenichkin et al., 2008), the most potent agonist in inducing PI hydrolysis through mGlu1a

receptors (Aramori and Nakanishi, 1992; Emery et al., 2010). These data suggest the existence of

ligand bias at mGlu1a receptors, where some agonists, such as quisqualate, can only stimulate

the G protein-mediated signaling, while others, such as glutamate, can induce both PI hydrolysis

and sustained ERK phosphorylation.

55

In order to further study the pharmacological properties of mGlu1a receptors without

interference from ionotropic or other metabotropic glutamate receptors, CHO cells stably

expressing mGlu1a receptors were used. As shown in Figure 21, all tested agonists stimulated PI

hydrolysis with quisqualate being the most potent agonist (EC50 ~ 1 μM), followed by glutamate

and DHPG (EC50 ~ 10 μM), and by aspartate and L-cysteic acid (EC50 ~ 100 μM). In addition to

the stimulation of PI hydrolysis, these agonists caused an increase in transient ERK

phosphorylation measured after five minutes of incubation with the agonist (Figure 22) which, as

shown previously, is PLC-dependent (Emery et al., 2010). The current data also indicate that the

transient ERK phosphorylation was induced by all mGlu1 agonists tested (Figure 22), with a

pharmacological profile and agonist potencies similar to those observed for PI hydrolysis (Figure

21). As expected, the mGlu1-selective noncompetitive antagonist YM-298198 (Kohara et al.,

2005) completely abolished both glutamate and quisqualate-induced ERK phosphorylation

(Figure 22).

The previous studies indicated that in addition to transient ERK phosphorylation, mGlu1a

receptors also cause a long-lasting β-arrestin1-dependent phosphorylation of ERK, which is

required for the mGlu1a-mediated protection from apoptosis (Emery et al., 2010). The sustained

ERK phosphorylation, measured after 24 hours, had a different pharmacological profile from

transient ERK phosphorylation (Figure 23), and was only induced by glutamate, aspartate and L-

cysteic acid, but not by quisqualate and DHPG (Figure 23). Glutamate-induced sustained ERK

phosphorylation was completely blocked by 10 μM YM-298198 (Figure 23).

56

Figure 21. Agonist profile for mGlu1a-mediated PI hydrolysis. Dose response curves of

agonist induced PI hydrolysis in CHO cells stably expressing mGlu1a receptors. Quisqualate

(Quis) was the most potent agonist followed by glutamate (Glu), DHPG, aspartate (Asp), and L-

cysteic acid (L-CA). Glutamate-induced PI hydrolysis was blocked by 10 M YM-298198

(YM). All data points are means from three independent experiments performed in triplicate with

error bars corresponding to S.E.M.

[Agonist], M

10-8 10-7 10-6 10-5 10-4 10-3

PI h

yd

roly

sis

(%

of

ba

sa

l)

100

200

300

400Glu

Quis

DHPG

Asp

L-CA

Glu+YM

57

[Agonist], M

10-6 10-5 10-4 10-3 10-2

Ph

osp

ho

ER

K (

% o

f b

asa

l)

100

200

300

400 GluQuisAspL-CADHPG

Glu+YMQuis+YM

Figure 22. Agonist profile for mGlu1a-mediated transient ERK phosphorylation. Dose

response curves of agonist induced ERK phosphorylation measured after 5 minutes of incubation

with ligands in CHO cells stably expressing mGlu1a receptors. Quisqualate (Quis) was the most

potent agonist followed by glutamate (Glu), aspartate (Asp), L-cysteic acid (L-CA), and DHPG.

Quisqualate and glutamate-induced ERK phosphorylation was blocked by 10 M YM-298198

(YM). All data points are means from three independent experiments performed in quadruplicate

with error bars corresponding to S.E.M.

58

[Agonist], M

10-5 10-4 10-3 10-2

Ph

osp

ho

ER

K (

% o

f b

asa

l)

100

150

200

250 GluQuisAspL-CADHPGGlu+YM

Figure 23. Agonist profile for mGlu1a-mediated sustained ERK phosphorylation. Dose

response curves of agonist induced ERK phosphorylation measured after 24 hours of incubation

with ligands in CHO cells stably expressing mGlu1a receptors. Glutamate (Glu) was the most

agonist, followed by aspartate (Asp), and L-cysteic acid (L-CA). Neither quisqualate nor DHPG

stimulated sustained ERK phosphorylation. Glutamate-induced ERK phosphorylation was

blocked by 10 M YM-298198 (YM). All data points are means from three independent

experiments performed in quadruplicate with error bars corresponding to S.E.M.

59

In addition, the potency and efficacy of the various mGlu1 agonists to elicit the protective

effect were tested. To this end, cells were transferred to serum-free culture medium to induce

apoptosis (Zhong et al., 1993) and their viability was assessed by MTT assays. Under these

conditions, after 3 days, cells exhibited approximately 30-40% of viability relative to controls

grown in serum-containing medium. The addition of glutamate, aspartate or L-cysteic acid to the

serum-free culture medium produced a substantial increase in viability, however in contrast to

the pharmacology of PI hydrolysis, quisqualate and DHPG were not effective in concentrations

up to 10 mM (Figure 24). The observed agonist potencies were similar to those observed for the

sustained phosphorylation of ERK through mGlu1a (Figures 23 and 24). The lack of effect by

quisqualate and DHPG is not likely due to agonist degradation or uptake since control

experiments were performed to test drug stability under cell culture conditions for the 3-day

incubation used in viability assays. Agonists were culture-conditioned with mGlu1a-expressing

CHO cells for 3 days, and then the collected media were used to stimulate PI hydrolysis in naïve

cells. As shown previously, both quisqualate and glutamate were equally potent at stimulating PI

hydrolysis before and after culture conditioning (Figure 7). Additionally, a similar bioassay of

DHPG showed no significant breakdown: EC50 = 30.6 + 4.7 μM for freshly-diluted DHPG, EC50

= 32.4 + 5.3 μM for preconditioned DHPG. These data indicate that the agonists tested are not

subject to degradation or uptake during the extended periods of incubation used.

60

[Agonist], M

10-5 10-4 10-3 10-2

Via

bility (

% o

f u

ntr

eate

d c

ontr

ol)

0

25

50

75

Glu

Quis

DHPG

Asp

L-CA

Figure 24. Agonist profile for mGlu1a-mediated protective signaling. Dose response curves

of agonist induced protection from serum deprivation in CHO cells stably expressing mGlu1a

receptors as measured by MTT assays. Glutamate (Glu) was the most agonist, followed by

aspartate (Asp), and L-cysteic acid (L-CA). Neither quisqualate (Quis) nor DHPG protected

cells. All data points are means from three independent experiments performed in triplicate with

error bars corresponding to S.E.M.

61

Taken together, these results confirm that PI hydrolysis, ERK phosphorylation, and the

protective signal transduction pathway are activated by the same receptors, but different mGlu1

receptor agonists appear to activate either one or both signal transduction pathways. Glutamate,

aspartate, and L-cysteic acid activate PI hydrolysis, transient and sustained ERK

phosphorylation, and protection; while quisqualate and DHPG only activate PI hydrolysis and a

transient phosphorylation of ERK. Such results can be explained by a phenomenon known as

ligand bias, which is the ability of some agonists, such as quisqualate, to activate only selected

signal transduction mechanisms while other agonists, such as glutamate, may activate all

signaling associated with the same receptor (Violin and Lefkowitz, 2007).

To further investigate mGlu1a receptor-mediated pharmacology and signal transduction, all

effects were measured in the presence of mGlu1-selective antagonists. To this end, both LY

367385 and 3-MATIDA, selective and competitive antagonists of mGlu1 receptors, (Clark et al.,

1997; Moroni et al., 2002) were used. As seen in Figures 25 and 26, LY 367385 and 3-MATIDA

both competitively inhibited glutamate-induced PI hydrolysis, causing the EC50 for glutamate to

shift from 17 + 2.2 μM to 101 + 15 μM in presence of LY 367385 (10 μM) and from 9.2 + 0.3

μM to 76 + 18 μM in the presence of 3-MATIDA (30 μM). Additionally, both competitive

antagonists were bioassayed after 3 days of culture conditioning and both were stable (Figures

25, 26). Competitive inhibition of glutamate-induced transient ERK phosphorylation was also

observed (Figure 27), causing the EC50 for glutamate to shift from 50 μM to 810 μM in presence

of LY 367385 (30 μM) and to 880 μM in presence of 3-MATIDA (30 μM).

62

[Glutamate], M

10-6 10-5 10-4 10-3

PI

Hyd

roly

sis

(%

of

ba

sa

l)

100

150

200

250 Control

10 M LY (Fresh)

10 M LY (CC)

Figure 25. Competitive antagonism of glutamate-induced PI hydrolysis by LY367685. Dose

response curves of glutamate-induced PI hydrolysis with competitive inhibition by freshly-

prepared (Fresh) and cultured-conditioned (CC) 10 μM LY367385 (LY). For glutamate, EC50 =

17 + 2.2 μM and 101 + 15 μM in the presence of either freshly-prepared or culture-conditioned

LY367385, demonstrating stability of this antagonist. Control refers to cells treated only with

indicated concentrations of glutamate and the solvent used to dilute LY367385. All data points

are means from three independent experiments performed in triplicate with error bars

corresponding to S.E.M.

63

Figure 26. Competitive antagonism of glutamate-induced PI hydrolysis by 3-MATIDA.

Dose response curves of glutamate-induced PI hydrolysis with competitive inhibition by freshly-

prepared (Fresh) and cultured-conditioned (CC) 30 μM 3-MATIDA. For glutamate, EC50 = 9.2 +

0.3 μM, and 76 + 18 μM in the presence of either freshly-prepared or culture-conditioned 3-

MATIDA, demonstrating stability of this antagonist. Control refers to cells treated only with

indicated concentrations of glutamate and the solvent used to dilute 3-MATIDA. All data points

are means from three independent experiments performed in triplicate with error bars

corresponding to S.E.M.

[Glutamate], M

10-6 10-5 10-4 10-3

PI

Hyd

roly

sis

(%

of

ba

sa

l)

100

150

200

250

Control

30 M 3-MATIDA (Fresh)

30 M 3-MATIDA (CC)

64

[Glutamate], M

10-5 10-4 10-3 10-2

Ph

osp

hoE

RK

(%

of ba

sal)

100

150

200

250

Contol

LY367385 (30 M)

3-MATIDA (30 M)

Figure 27. Competitive antagonism of glutamate-induced transient ERK phosphorylation.

Dose response curves of glutamate-induced transient ERK phosphorylation with competitive

inhibition by 30 μM LY367385 or 30 μM 3-MATIDA. For glutamate, EC50 = 50 μM, shifting to

810 μM in presence of LY 367385 (30 μM) and to 880 μM in presence of 3-MATIDA (30 μM).

All data points are means from three independent experiments performed in triplicate with error

bars corresponding to S.E.M.

65

In contrast, even high concentrations (1 mM) of either LY 367385 or 3-MATIDA failed to

inhibit glutamate-induced sustained (24 hour) ERK phosphorylation (Figure 28), where the EC50

for glutamate did not vary significantly from 301 μM in the presence or absence of either

competitive antagonist. However, glutamate-induced sustained ERK phosphorylation was

completely inhibited by 30 μM YM-298198, an mGlu1-selective noncompetitive antagonist

(Kohara et al., 2005). As in the case of glutamate-induced sustained ERK phosphorylation, the

addition of either LY 367385 (1 mM) or 3-MATIDA (1 mM) had no effect on glutamate-induced

protection (Figure 29). Instead, the protective signaling was noncompetitively inhibited by YM-

298198 (3 μM), confirming that this effect is mediated by mGlu1 receptors.

Because sustained ERK phosphorylation and protective signaling were inhibited by

noncompetitive antagonists, but not by the competitive antagonists LY 367285 and 3-MATIDA,

we hypothesized that glutamate may bind to different sites to stimulate PI hydrolysis and to

induce protective signaling. Previously, the X-ray crystallography of the mGlu1 binding site with

bound glutamate has been resolved (Kunishima et al., 2000). These studies, as well as mutational

analyses of the mGlu1 binding site (Sato et al., 2003), indicate that several amino acid residues,

including Thr188

, Asp208

, Tyr236

, and Asp318

, interact with the amino group of the glutamate and

these interactions are necessary for receptor activation as measured by PI hydrolysis and the

binding of quisqualate (Sato et al., 2003). In contrast, there are additional residues in the binding

site, including Arg323

and Lys409

, which are postulated to bind the omega-carboxyl group of

glutamate, but mutation of these residues does not attenuate glutamate-induced PI hydrolysis or

the binding of quisqualate (Sato et al., 2003).

66

Figure 28. Lack of competitive antagonism on glutamate-induced sustained ERK

phosphorylation. Dose response curves of glutamate-induced sustained (24 hour) ERK

phosphorylation in the presence of competitive antagonists by LY367385 (1 mM) or 3-MATIDA

(1 mM). For glutamate, EC50 = 300 μM, neither competitive antagonist changed the potency of

glutamate. Glutamate-induced sustained ERK phosphorylation was effectively blocked by the

noncompetitive antagonist YM-298198 (30 μM). All data points are means from three

independent experiments performed in triplicate with error bars corresponding to S.E.M.

[Glutamate], M

10-4 10-3 10-2

Ph

osp

ho

ER

K (

% o

f b

asa

l)

100

150

200

Control

LY367385 (1 mM)

3-MATIDA (1 mM)

YM298198 (30 M)

67

[Glutamate], M

10-5 10-4 10-3

Via

bili

ty

(% o

f untr

eate

d c

ontr

ol)

40

60

80

Control

LY367385 (1 mM)

3-MATIDA (1 mM)

YM298198 (3 M)

Figure 29. Lack of competitive antagonism on glutamate-induced protective signaling. Dose

response curves of glutamate-induced protective signaling in the presence of competitive

antagonists by LY367385 (1 mM) or 3-MATIDA (1 mM). Neither competitive antagonist changed

the potency of glutamate, however glutamate-induced protection was noncompetitively inhibited

by the noncompetitive antagonist YM-298198 (3 μM). All data points are means from four

independent experiments performed in triplicate with error bars corresponding to S.E.M.

68

In order to ascertain the mechanism by which different ligands can activate different signal

transduction pathways through the same receptor, we have mutated Thr188

, Arg323

and Lys409

residues of the mGlu1a ligand binding site, selected based on X-ray crystallographic studies of

the mGlu1 ligand binding domain (Kunishima et al., 2000).

All mutated constructs of mGlu1a were stably expressed in CHO cells, and in the case of

all mutants protein expression levels were comparable to wild-type mGlu1a receptors (Figure

30). The mGlu1a construct with the T188A, mutation which has been shown to be critical for the

binding of quisqualate and activation of PI hydrolysis (Sato et al., 2003) was, as expected, unable

to enhance PI hydrolysis, as the observed response was not different from that obtained with

CHO cells transfected with the control empty vector (Figure 31). These data confirm that the

Thr188

residue is required for classical PI-linked signaling through mGlu1a receptors. In contrast,

the T188A mutation failed to block the ability of mGlu1 to induce the β-arrestin1-mediated

sustained ERK phosphorylation and protective signaling (Figures 33, 34). For additional controls

to ascertain the role of β-arrestin-1, the expression of β-arrestin-1 was silenced by shRNA in

CHO cells stably transfected with mGlu1a T188A mutant. As seen in Figure 32, shRNA targeted

to β-arrestin-1 caused an approximately 70% reduction in β-arrestin-1 expression. Cells

expressing shRNA against β-arrestin-1 were then transfected with human β-arrestin-1, which is

refractory to shRNA due to several mismatched bases. Expression of human β-arrestin-1 caused

a substantial increase in protein expression of β-arrestin-1 (Figure 32). While glutamate

stimulated both sustained ERK phosphorylation (Figure 33) and protective signaling (Figure 34),

the silencing of β-arrestin-1 with shRNA resulted in the blockade of both responses.

69

Figure 30. Protein expression levels of mutant mGlu1a receptors. Representative Western

blot depicting protein expression levels of mutated mGlu1a receptors stably transfected in CHO

cells. All receptors were expressed at equivalent levels to wild-type (W/T) control mGlu1a

receptors.

70

10-6 10-5 10-4

PI H

yd

roly

sis

(%

of b

asa

l)

100

150

200

250

mGlu1

Vector

T188A

[Glutamate], M

Figure 31. Lack of classical mGlu1a receptor signaling in T188A mutant receptors.

Glutamate-induced PI hydrolysis occurs to a similar extent in CHO cells transfected with vector

(pcDNA 3.1) and CHO cells stably expressing the T188A mutant mGlu1a receptor. Data points

are means from three independent experiments performed in quadruplicate with error bars

representing S.E.M.

71

Figure 32. Silencing of -arrestin-1 in CHO cells expressing T188A mutant mGlu1a

receptors. Representative Western blot depicting efficient silencing of -arrestin-1 by shRNA.

Protein expression levels were restored by transfection with human β-arrestin-1.

72

[Glutamate], M

10-5 10-4 10-3 10-2

ph

osph

oE

RK

(%

of ba

sa

l)

50

100

150

200

250

300

350T188A + Scrambled shRNA

T188A + shRNA

T188A + shRNA + Arr1

Figure 33. -arrestin-1 dependent sustained ERK phosphorylation in T188A mutant

mGlu1a receptors. In T188A mutant mGlu1a receptors stably expressed in CHO cells,

glutamate-induced ERK phosphorylation measured after 24 hours of agonist stimulation is

blocked by shRNA silencing of β-arrestin-1 and is restored by overexpression of human β-

arrestin-1. All data points are means from three experiments performed in quadruplicate with

error bars representing S.E.M.

73

Figure 34. Glutamate-induced protective signaling in T188A mutant mGlu1a receptors.

Glutamate-induced protective signaling through T188A mutant mGlu1a receptors expressed in

CHO cells is inhibited by shRNA silencing of β-arrestin-1. Glutamate-induced protection is

restored upon overexpression of human β-arrestin-1. All values are means from at least three

independent experiments performed in triplicate with error bars representing S.E.M.

[Glutamate], M

10-5 10-4 10-3 10-2

Via

bili

ty

(% o

f U

ntr

ea

ted

Con

tro

l)

40

50

60

70

80

90

100T188A + Scrambled shRNA

T188A + shRNA

T188A + shRNA + Arr1

74

However, these responses were restored when the β-arrestin-1-depleted cells were rescued by

transfection with human β-arrestin-1 (Figures 33, 34). Taken together, these data indicate that the

interaction of glutamate with the Thr188

residue is required for the coupling with PI hydrolysis,

but is not necessary for the coupling with sustained ERK phosphorylation and protective

signaling.

Because Thr188

has been shown to interact with the alpha amino group of mGlu1 agonists

(Sato et al., 2003), the next hypothesis was that sustained ERK phosphorylation and protective

signaling require ligand interactions with different residues of the binding site. Therefore,

mutations were performed on residues Arg323

and Lys409

, which are postulated to interact with

the omega carboxyl group of glutamate, but do not functionally interact with quisqualate (Sato et

al., 2003). In CHO cells stably transfected with either R323V or K409A mutant of mGlu1a,

agonist-induced PI hydrolysis was equivalent to cells expressing wild-type mGlu1a receptors

(Figure 35). These data are consistent with a previous report indicating that these residues are not

needed for quisqualate binding or PI hydrolysis (Sato et al., 2003). As expected, glutamate

stimulation of both mutated mGlu1a receptors also caused an increase in PLC-dependent,

transient ERK phosphorylation (Figure 36), which was abolished by the PLC inhibitor U73122

(Figure 36). In contrast, both R323V and K409A mGlu1a mutants, expressed in CHO cells,

failed to stimulate the sustained ERK phosphorylation (Figure 37), and failed to protect against

serum withdrawal-induced apoptosis (Figure 38).

75

[Glutamate], M

10-6 10-5 10-4 10-3

PI

Hyd

roly

sis

(%

ba

sa

l)

100

150

200

250

300

350mGlu1

R323V

K409A

Figure 35. Classical signaling in R323V and K409A mutant mGlu1a receptors. In both

R323V and K409A mutant mGlu1a receptors expressed in CHO cells, glutamate-induced PI

hydrolysis occurs to a similar extent as in CHO cells expressing the wild-type mGlu1a receptors

(EC50 = 18 μM, 16 μM , and 13 μM, respectively). All data points are means from three

independent experiments performed in triplicate with error bars representing S.E.M.

76

Figure 36. ERK phosphorylation is G protein-dependent in R323V and K409A mutant

mGlu1a receptors. Glutamate-induced ERK phosphorylation measured after 5 minutes of

agonist stimulation was completely inhibited by PLC inhibitor U73122 (U73), suggesting ERK

phosphorylation through R323V and K409A mutant mGlu1a receptors is completely G protein-

dependent. All data points are means from two experiments performed with six replications and

error bars represent S.E.M.

[Glutamate], M

10-6 10-5 10-4 10-3

Ph

osp

hoE

RK

(%

of b

asa

l)

100

150

200

250

300

R323V

R323V + U73 (30 M)

K409A

K409A + U73 (30 M)

77

Figure 37. R323V and K409A mutant mGlu1a receptors do not stimulate long-term ERK

phosphorylation. Glutamate-induced ERK phosphorylation measured after 24 hours of agonist

stimulation was not significantly elevated over basal in R323V or K409A mutant mGlu1a

receptors expressed in CHO cells. All data points are means from two experiments performed

with six replications and error bars represent S.E.M.

W/T mGlu1a R323V K409A

Ph

osp

ho

ER

K (

% b

asa

l)

100

150

200

250 10 mM Glutamate

78

Figure 38. R323V and K409A mutant mGlu1a receptors do not protect CHO cells.

Glutamate-induced protection from serum deprivation-induced toxicity was measured after three

days agonist stimulation by MTT assays. Glutamate failed to increase the viability of CHO cells

stably expressing R323V or K409A mutant mGlu1a receptors. All data points are means from

three experiments performed with six replications and error bars represent S.E.M.

[Glutamate], M

10-5 10-4 10-3 10-2

Via

bili

ty

(% o

f u

ntr

ea

ted

co

ntr

ol)

40

60

80

mGlu1

R323V

K409A

79

Based on the data suggesting that interaction with residues Arg323

and Lys409

, which are

thought to bind the omega carboxyl group of glutamate, are not necessary for agonist-induced PI

hydrolysis but, instead, are required for receptor-mediated protective signaling, we reasoned that

a ligand able to interact with Arg323

and Lys409

, but not with Thr188

should induce protection, but

not PI hydrolysis when applied to wild-type mGlu1a receptors. Such a ligand, glutaric acid, is an

analog of glutamate that lacks the alpha amino group, which interacts with Thr188

, therefore it

would not be expected to stimulate PI hydrolysis. Similarly, succinic acid is an analog of

aspartate which also lacks an alpha amino group. In fact, in CHO cells stably expressing mGlu1a

receptors, glutaric and succinic acids both failed to enhance PI hydrolysis, even at very high

concentrations (Figure 39), suggesting that they are not classical mGlu1a agonists. In contrast,

glutaric acid stimulated, in a dose-dependent manner, both the sustained ERK phosphorylation

(Figure 40) and the protective signaling of mGlu1 receptors (Figure 41). Moreover, glutaric acid

protected cerebellar granule cells, which express native mGlu1a receptors, from low potassium-

induced apoptosis in a dose-dependent manner (Figure 42). Succinic acid protected cells from

apoptosis to a similar extent (Figure 43). The effects of glutaric and succinic acid were blocked

by the noncompetitive mGlu1 antagonist YM-298198, confirming their site of action at this

receptor (Figures 41-43).

80

Figure 39. Effects of glutaric and succinic acids on PI hydrolysis. Neither glutaric nor

succinic acid caused an elevation in PI hydrolysis in CHO cells stably expressing mGlu1a

receptors. Glutamate (1 mM) served as a positive control in these experiments. Values are means

from three experiments performed in quadruplicate and error bars represent S.E.M.

PI H

yd

roly

sis

(%

of b

asa

l)

100

150

200

250

300

350

Glutaric Acid

Glutamate

Succinic Acid

1 mM 3 mM 10 mM

81

[Glutaric Acid], M

10-5 10-4 10-3 10-2

Pho

sp

ho

ER

K (

% o

f b

asa

l)

100

150

200

250

5 min

5 min + YM (10 M)

24 h.

24 h. + YM (10 M)

Figure 40. Glutaric acid is an mGlu1a receptor agonist that causes ERK phosphorylation.

ERK phosphorylation in CHO cells expressing mGlu1a receptors was measured after 5 minutes

and 24 hours of incubation with glutaric acid increased in a dose-dependent manner. All effects

of glutaric acid were noncompetitively inhibited by YM-298198 (YM). All data points are means

from three experiments performed in triplicate with error bars representing S.E.M.

82

Figure 41. Glutaric acid protects CHO cells through activity at mGlu1a receptors. Dose-

dependent effect of glutaric acid on protecting CHO cells expressing mGlu1a receptors from

serum deprivation-induced toxicity. Glutaric acid was noncompetitively inhibited by YM-

298198 (YM). All data points are means from four experiments performed in triplicate with error

bars representing S.E.M.

[Glutaric Acid], M

10-5 10-4 10-3 10-2

Via

bili

ty

(% o

f u

ntr

ea

ted

co

ntr

ol)

50

75

100

YM (10 M)

83

Figure 42. Glutaric acid protects cerebellar granule neurons through mGlu1a receptors.

Dose-dependent effect of glutaric acid (GA) on viability of cerebellar granule neurons subjected

to 5 mM potassium (K5) overnight to induce apoptosis. The effect of glutaric acid was

noncompetitively inhibited by 10 M YM-298198 (YM). Viability was measured by MTT assay.

All data points were normalized to controls grown in 25 mM potassium (K25) and are means

from four experiments performed in triplicate with error bars representing S.E.M.

Glutaric Acid, [M]

10-6 10-5 10-4 10-3 10-2

Via

bility (

% o

f K

25

Co

ntr

ol)

40

50

60

70

80

90

GA

GA + YM298198

84

Figure 43. Succinic acid protects CHO cells through activity at mGlu1a receptors. Dose-

dependent effect of succinic acid on protecting CHO cells expressing mGlu1a receptors from

serum deprivation-induced toxicity. Succinic acid was noncompetitively inhibited by YM-

298198 (YM). All data points are means from four experiments performed in triplicate with error

bars representing S.E.M.

[Succinic Acid], M

10-5 10-4 10-3 10-2

Via

bili

ty

(% o

f untr

eate

d c

ontr

ol)

50

75

100

YM (10 M)

85

The ability of the competitive antagonists LY 367385 and 3-MATIDA to inhibit only PI

hydrolysis and transient ERK phosphorylation, but not the sustained ERK phosphorylation or

protective signaling, suggests that glutamate may bind to two distinct sites to stimulate the two

different signaling cascades. If these sites were truly separate, then ligands binding to one site

(site A) should not interfere with the potency of agonists binding to the second site (site B). This

possibility was investigated by testing for interactions between the biased ligands.

First, the ability of quisqualate (biased ligand for site A) to interfere with the potency of

glutamate to induce protective signaling was tested, and no effect was found, even at extreme

(300 μM) concentrations of quisqualate (Figure 44). Second, we tested the ability of glutaric acid

(biased ligand at site B) to interfere with glutamate-induced PI hydrolysis, and found it equally

inactive at concentrations up to 10 mM (Figure 45). These data strongly suggest that mGlu1a

receptors possess two distinct, non-interacting glutamate binding sites, and the binding of

agonists to each site allows for the stimulation of a different signal transduction cascade.

86

Figure 44. Quisqualate fails to inhibit glutamate-induced protection at mGlu1a receptors.

Glutamate-induced protection from serum deprivation-induced toxicity in CHO cells expressing

mGlu1a receptors in the presence of varying concentrations of quisqualate. In the presence of

quisqualate, the EC50 for glutamate to protect did not vary significantly from 320 μM. All data

points are means from five experiments performed in triplicate with error bars representing

S.E.M.

[Glutamate], M

10-5 10-4 10-3 10-2

Via

bili

ty

(% o

f u

ntr

ea

ted

co

ntr

ol)

40

60

80

100

Quis = 0 M

Quis = 30 M

Quis = 300 M

87

Figure 45. Glutaric acid fails to inhibit glutamate-induced PI hydrolysis at mGlu1a

receptors. Glutamate-induced PI hydrolysis in CHO cells expressing mGlu1a receptors in the

presence of varying concentrations of glutaric acid. In the presence of glutaric acid, the EC50 for

glutamate to protect did not vary significantly from 13 μM. All data points are means from three

experiments performed in triplicate with error bars representing S.E.M.

[Glutamate], M

10-6 10-5 10-4 10-3

PI H

yd

roly

sis

(%

ba

sa

l)

100

150

200

250

300

350GA = 0

GA = 1 mM

GA = 3 mM

GA = 10 mM

88

DISCUSSION

Previous studies with mGlu1a receptors have revealed some unusual properties of these

receptors. As shown in primary cultures of cerebellar and cortical neurons, increased mGlu1a

receptor expression led to apoptotic cell death (Pshenichkin et al., 2008). This toxic effect was

not blocked by mGlu1 antagonists but by silencing receptor expression. Unexpectedly, mGlu1a

toxicity was also blocked by its agonist glutamate. These properties allow us to categorize

mGlu1a as a dependence receptor, defined as a receptor which, when expressed, promotes

apoptosis in the absence of its ligand, but stimulates survival in its presence (Mehlen and

Bredesen, 2004). Hence, mGlu1a receptors would make the survival of neuronal cells

“dependent” on the presence of the endogenous agonist glutamate. While the negative signaling

of mGlu1a which leads to apoptosis still remains to be elucidated, the positive signaling has been

described in our previous studies (Emery et al., 2010; Pshenichkin et al., 2008). This positive,

protective signaling is not mediated by the classical G protein- mediated coupling of mGlu1a

receptors, but, instead, involves a β-arrestin-1-dependent internalization of mGlu1a, followed by

the stimulation of the MEK/ERK pathway (Emery et al., 2010). However, the protective

signaling of mGlu1a showed a different agonist profile than the G protein-mediated stimulation

of PLC.

In this study, those different pharmacological profiles of the two mGlu1a responses were

investigated and revealed the existence of a ligand bias at these receptors. These studies indicate

the existence of three classes of agonists at mGlu1a receptors: (1) unbiased ligands, such as

glutamate, aspartate, and cysteic acid, which activate both G protein-dependent signaling and β-

arrestin-dependent protective signaling, (2) ligands biased towards G protein signaling, such as

89

quisqualate and DHPG, and (3) previously unknown ligands biased towards β-arrestin-dependent

signaling, such as glutaric or succinic acid (Table 2). This is the first report of ligand bias at a

metabotropic glutamate receptor, but these findings should come as no surprise, as ligand bias

has been described at numerous other GPCRs, as reviewed in (Violin and Lefkowitz, 2007).

The initial clue as to the mechanism responsible for ligand bias at mGlu1a receptors came

from results indicating that, in contrast to PI hydrolysis, the protective signaling was not

inhibited by competitive mGlu1 antagonists, while both responses were blocked by

noncompetitive mGlu1 antagonists. This would be explained if glutamate induced protective

signaling by binding to a site distinct from that occupied by competitive antagonists. Instead,

noncompetitive antagonists would still inhibit both responses, possibly by modifying receptor

conformation. In fact the mGlu1-selective competitive antagonists LY 367385 and 3-MATIDA,

which both inhibit glutamate-induced PI hydrolysis and transient ERK phosphorylation, had no

effect on glutamate-induced sustained ERK phosphorylation or protective signaling at

concentrations as high as 1 mM. In contrast, all mGlu1a signaling was inhibited by the mGlu1-

selective noncompetitive antagonist YM 298198, which binds to an allosteric site situated on the

7th

transmembrane domain of the receptor (Kohara et al., 2005). Previously, similar results were

obtained using other mGlu1-selective noncompetitive antagonists, including CPCCOEt and

JNJ16259685 (Emery et al., 2010), indicating that all effects are selectively mediated by

mGlu1a, but not necessarily due to interactions with the same orthosteric binding site which also

binds competitive antagonists (Clark et al., 1997).

90

Agonist Type of agonism Wild type mGlu1a

T188A mutant

R323V & K409A mutants

Glutamate, Aspartate, Cysteate

Unbiased PI / PR -- / PR PI / --

Quisqualate, DHPG G protein-biased PI / -- -- / -- PI / --

Glutarate, Succinate β-arrestin-biased -- / PR -- / PR -- / --

Table 2. Different responses induced by three classes of agonists acting at wild type and

mutated mGlu1a receptors. PI: PI hydrolysis, PR: protection from apoptosis

91

The hypothesis for the existence of a second glutamate binding site on mGlu1a receptors is

further supported by the current mutational studies the T188A mutation on mGlu1a receptors

results in a complete block of signaling through G protein-dependent pathways, however

signaling which results in sustained ERK phosphorylation and protection from apoptosis through

the β-arrestin-1-dependent mechanism remains active. This indicates that the Thr188

residue is

required for classical signaling through mGlu1a receptors, but an interaction with this residue is

not necessary for β-arrestin-1-dependent signaling. Conversely, R323V and K409A mutants of

mGlu1a receptors do not induce β-arrestin-1-dependent, sustained, ERK phosphorylation or

protective signaling, but still enhance PI hydrolysis in the presence of glutamate to a similar

extent as wild-type mGlu1a receptors. This indicates that glutamate interaction with Arg323

and

Lys409

is needed for β-arrestin-1-dependent signaling. These mutational data are consistent with a

previous mutational analysis of the ligand binding domain of mGlu1a receptors, however β-

arrestin-dependent signaling was not tested in these studies (Sato et al., 2003).

Strong evidence for the existence of two distinct glutamate binding sites comes from the

study of interactions between the biased and unbiased mGlu1 ligands. Quisqualate, the most

potent ligand of mGlu1 receptors, biased towards G protein-mediated signaling, failed to inhibit

glutamate-induced protective signaling, even at very high concentrations. Similarly, glutaric

acid, an agonist biased towards protective signaling, failed to change the potency of glutamate to

stimulate PI hydrolysis. This lack of interactions indicates that biased ligands likely bind to two

distinct, non-interacting sites. While novel, these data suggesting the presence of two separate

glutamate binding sites on mGlu1a receptors are in keeping with some recent studies on mGlu

receptors. In a study of mGlu receptor homology, an allosteric ion binding site was located

92

adjacent to the orthosteric glutamate binding site (Ogawa et al., 2010). Interestingly, a ligand has

since been discovered which simultaneously interacts with the orthosteric glutamate site and

newly discovered allosteric site in mGlu4 receptors (Acher et al., 2011), suggesting the

possibility of multiple agonist binding motifs in other mGlu receptors. In case of mGlu1, our

mutational data indicate that the two binding sites, while separate, are still located in the same

general region of the receptor N-terminal domain. If confirmed, this would require some

revisions to the conformational model of the receptor N-terminal domain. The current model

assumes that Thr188

, as well as Arg323

and Lys409

residues interact with a single molecule of

glutamate. Such a conformation would not sterically allow for a simultaneous non-interacting

binding of two glutamate molecules within the proposed binding pocket. An alternative

possibility is the location of the two binding sites on separate subunits of the mGlu1a receptor

homodimer, however, we are not aware of any data supporting such a hypothesis.

In the present study, new ligands for mGlu1a receptors which are biased towards the

protective signaling were identified. They are glutaric and succinic acids, analogues of glutamate

and aspartate, respectively, with a deleted alpha-amino group. Other homologues of these

dicarboxylic acids, with shorter (oxalic acid and malonic acid) or longer (adipic acid, and pimelic

acid) carbon chains were inactive (data not shown). Consistent with the current findings of

cytoprotective properties, succinate has previously been shown to ameliorate cognitive defects in

a rat model of Alzheimer’s disease (Storozheva et al., 2008). While both glutarate and succinate

are endogenous to the brain, they are common metabolites, and it is unclear whether they could

serve a physiological role, acting at native mGlu1 receptors. However, these compounds may

now be used as lead structures in the design of new biased agonists, and especially antagonists,

93

with ability to affect mGlu1 receptor-mediated protective signaling, without disturbing the

classical G protein-mediated signal transduction.

94

CHAPTER 4

Thesis Conclusion

95

Major findings of this thesis

The existence of a new signal transduction mechanism of mGlu1a receptors is

demonstrated in Chapter 2. In contrast to the classical G protein-mediated PI hydrolysis, this

mechanism appears to involve agonist-mediated, β-arrestin-1-dependent receptor internalization,

followed by the activation of MEK cascade and a long-lasting, sustained phase of ERK

phosphorylation. This mechanism appears to be responsible for the protective action of

glutamate mediated by mGlu1a receptors. These experiments were described in the first

published report of this signal transduction pathway though a metabotropic glutamate receptor

(Emery et al., 2010). While novel, these findings should come as no surprise, since β-arrestin-1-

dependent sustained ERK phosphorylation has previously been demonstrated through numerous

other G protein-coupled receptors (Broca et al., 2009; Quan et al., 2008; Zheng et al., 2008).

Previously, it was proposed that mGlu1a functions as a dependence receptor causing

apoptosis in the absence of glutamate, possibly due to the cleavage of its C-terminal domain

(Pshenichkin et al., 2008). Now we propose that the protective, positive signaling of mGlu1,

which occurs in the presence of glutamate, is mediated by this new, G protein-independent,

mechanism of signal transduction. This hypothesis needs now to be more extensively validated

in systems expressing native mGlu1a receptors and in vivo. Further studies are also needed to

address the mechanism and establish the conditions in which glutamate, classically viewed as an

excitotoxin (Bruno et al., 1995; Siliprandi et al., 1992), may also produce a protective effect

when acting at mGlu1 receptors. Acting as a dependence receptor, mGlu1a may serve as a sensor

of extracellular glutamate, promoting neuronal survival in the presence of glutamate and

inducing apoptosis in its absence. Such a mechanism could play an important role in brain

96

physiology by allowing glutamate to act as a trophic factor contributing to neuronal development

and neuronal selection during synaptogenesis and, possibly, by participating in the restructuring

of damaged brain tissue.

In Chapter 3, the molecular mechanism for ligand bias at mGlu1 receptors was

investigated. Multiple experiments indicated that some agonists, such as glutamate, aspartate,

and L-cysteic acid, stimulate both PI hydrolysis and protective signaling through mGlu1a

receptors while other agonists, such as quisqualate and DHPG, only stimulate PI hydrolysis.

Mutations of selected residues of the ligand binding domain of the receptor revealed that Thr188

is necessary for PI hydrolysis while Arg323

and Lys409

are necessary for protective signaling but

not PI hydrolysis. These data suggest that the interaction of the -amino group of a ligand and

the receptor is required for PI hydrolysis while interaction between the omega-carboxyl group of

the ligand and the receptor is necessary for protective signaling. This hypothesis was supported

by the fact that glutaric and succinic acids, analogs of glutamate and aspartate lacking the -

amino group, stimulated sustained ERK phosphorylation and protective signaling selectively

through mGlu1a receptors. This is the first report of these two compounds as agonists at any

mGlu receptor. Finally, glutamate-induced protection was not inhibited by quisqualate, even at

very high concentrations. Likewise, glutaric acid did not inhibit glutamate-induced PI hydrolysis.

Taken together, these data suggest the existence of two distinct non-overlapping glutamate

binding sites on mGlu1a receptors, with agonist action at one site (Site A) being responsible for

G protein-dependent signaling and agonist action at the second site (Site B) being responsible for

sustained ERK phosphorylation and protective signaling.

97

Future directions

Many elements of the protective signaling cascade initiated by mGlu1a receptors remain

unknown. Future studies should investigate the hypothesis that the C-terminus of mGlu1a

receptors is phosphorylated with GRK4 prior to the binding of -arrestin-1. The protective

substrates of phosphorylated ERK remain unknown. Since phosphorylated ERK that is linked to

-arrestin generally acts upon cytosolic substrates, the phosphoproteins should be investigated

using antibodies. Moreover, the changes in gene regulation that result from this signaling

cascade could be investigated through the use of microarrays and further investigated using

pharmacological approaches. Such studies could be useful in identifying other potential methods

to protect cells from toxicity. Additionally, these studies should be more comprehensively

confirmed in primary cultures of neurons and ultimately in vivo. Such studies could be very

useful in identifying a role for glutamate to act as a trophic factor in the developing brain, and

possibly as a neuroprotective mechanism. Additionally, emerging evidence suggest that

activation of mGlu1 receptors plays a role in the proliferation of melanoma cells (Marin and

Chen, 2004). Further studies should test the hypothesis that -arrestin-dependent signaling

through mGlu1 receptors is responsible for this increase in proliferation. If confirmed, these

results would yield multiple additional molecular targets for the treatment of melanoma.

The studies in Chapter 3 represent interesting possibilities for further studies. Taken

together, these results suggest the existence of two glutamate binding sites at mGlu1a receptors.

A more thorough mutational analysis of the ligand binding domain of the receptor paired with

computer modeling may lead greater insight into the second binding site. Pharmacological

studies indicate that glutamate, aspartate, and L-cysteic acid act at Site B, and glutaric and

98

succinic acids were identified as biased agonists for this site on mGlu1a receptor. Neither of

these newly-identified compounds is very potent, but they may serve as lead compounds for

further rational drug design, as ligands which do not stimulate PI hydrolysis but do stimulate

protective signaling could be quite useful for possible use in neurodegeneration. Moreover, since

mGlu1 receptor activation increases the proliferation of melanoma cells (Marin and Chen, 2004),

identification of a compound that selectively antagonizes Site B could be extremely useful, since

it could potentially be used to inhibit the proliferation of melanoma cells while not affecting G

protein-dependent mGlu1 receptor signaling, which occurs due to agonist activity at Site A.

99

Construct Sequence

T188A Forward

5’CAGATCGCCTATTCTGCCGCTAGCA

Reverse 5’GTCACTCAGGTCTATGCTAGCGGGA

R323V Forward 5’GAAGTGATGGATGGGCAGACGTCGACGAAGTCATCGAAGGC

Reverse 5’GCCTTCGATTTCGTCGACGTCTGCCCATCCATCACTTC

K409A Forward 5’GAAAACTATGTCCAGGACAGCGCCATGGGATTTGTCATCAATGCC

Reverse 5’GGCATTGATGACAAATCCCATCGGGCTGTCCTGGACATAGTTTTC

Table 3. Sequences of PCR primers for creating mutant mGlu1 receptors

100

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