Michael F. RobertsAnne E. Kruchten

Receptor Biology

Michael F. Roberts and Anne E. Kruchten

Receptor Biology

Authors

Michael F. RobertsLinfield CollegeBiology DepartmentMcMinnville97128 Murdock 216 ORUnited States

Anne E. KruchtenLinfield CollegeBiology Department900 SE Baker Street97128 McMinnville ORUnited States

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To our mentors: Warren Porter, University of Wisconsin – Madison and David Bernlohr,University of Minnesota.

To our families:

Mike Roberts Mike Kruchten

LuisJohn PaulAmeliaRosemary

Yarrow

Christopher

Sherill Anne

VII

Contents

Acknowledgment XIII

Part I Introduction 1

1 Introduction 31.1 Receptors and Signaling 31.1.1 General Aspects of Signaling 31.1.2 Verbal and Physiological Signals 31.1.3 Criteria for Recognizing Transmitters

and Receptors 41.1.4 Agonists 41.1.5 Receptors 41.1.6 Receptor–Enzyme Similarities 41.2 Types of Receptors and Hormones 51.2.1 Receptor Superfamilies 51.3 Receptors Are the Chemical Expression

of Reality 6

2 The Origins of Chemical Thinking 92.1 Overview of Early Pharmacological

History 92.1.1 The Development of a Chemical

Hypothesis 92.1.2 Chemical Structure and Drug Action 102.1.3 The Site of Drug Action 102.2 Modern Pharmacology 102.2.1 Langley and Ehrlich: the Origins of the

Receptor Concept 102.2.2 Maturation of the Receptor Concept 132.3 Phylogenetics of Signaling 132.3.1 The First Communicators 13

Part II Fundamentals 15

3 Membranes and Proteins 173.1 Membranes 173.1.1 The Cytoplasmic Membrane – the

Importance of Cell Membranes 173.1.2 History of Membrane Models 173.1.2.1 The Roles of Proteins in Membranes 183.1.2.2 Challenges to the Danielli–Davson

Model 19

3.1.2.3 A New View of Membrane Proteins 193.1.2.4 The Modern Concept of

Membranes – the Fluid MosaicModel 19

3.1.3 Membrane Components 193.1.3.1 Membrane Lipids 193.1.3.2 Asymmetry and Heterogeneity in

Membrane Lipids 203.1.3.3 Membrane Construction and Insertion of

Proteins 203.2 The Nature and Function of Proteins 213.2.1 Linear and Three-Dimensional

Structures 223.2.2 Primary Structure 223.2.3 Secondary Structure 233.2.4 Tertiary Structure 243.2.5 Protein Domains 253.2.6 Proteomics 25

4 Hormones as First Messengers 274.1 Hormones and Cellular

Communication 274.1.1 Discovery of Hormones 274.2 Types of Hormones 274.2.1 Pheromones for Signaling between

Individuals 284.2.2 Archaea and Bacteria 284.2.3 Eukaryotes 294.2.3.1 Chromalveolates 294.2.3.2 Unikonts – Amoebozoa, Fungi,

Animals 294.2.3.3 Invertebrate Pheromones 314.2.3.4 Vertebrate Pheromones 314.3 Vertebrate Hormones and

Transmitters 314.3.1 Peptide and Non-Peptide Agonists 314.3.1.1 Peptides 314.3.1.2 Non-peptides 314.3.2 Peptide Hormones of the

G-Protein-Coupled Receptors 324.3.2.1 Hypothalamic-Pituitary Axis 32

VIII Contents

4.3.2.2 The Anterior Pituitary TrophicHormones 34

4.3.3 Other Neural Peptides 354.3.3.1 Opioids 354.3.3.2 Non-Opioid Transmitter Peptides 364.3.4 Peptides from Non-Neural Sources 364.3.4.1 Digestive Tract Hormones 364.3.4.2 Hormones from Vascular Tissue 384.3.4.3 Hormones from the Blood 384.3.4.4 Peptide Hormones from Reproductive

Tissues 394.3.4.5 Hormones from Other Tissues 394.3.5 Non-Peptides Acting on

G-Protein-Coupled Receptors 394.3.5.1 Transmitters Derived from Amino

Acids 394.3.5.2 Transmitters Derived from

Nucleotides 404.3.5.3 Transmitters Derived from Membrane

Lipids – Prostaglandins andCannabinoids 41

4.3.6 Transmitters of the Ion Channels 414.3.7 Hormones of the Receptor

Kinases – Growth Factor Receptors 434.3.7.1 Insulin 434.3.7.2 Insulin-Like Growth Factors 434.3.7.3 Natriuretic Peptides 434.3.7.4 Peptide Signal Molecules Important in

Embryogenesis 434.3.7.5 Pituitary Gland

Hormones – Somatotropin andProlactin 43

4.3.8 Hormones of the Nuclear Receptors 444.3.8.1 Steroids 444.3.8.2 Non-Steroid Nuclear Hormones 464.4 Analgesics and Venoms as Receptor

Ligands 46

5 Receptor Theory 475.1 The Materialization of Receptors 475.2 Receptor Mechanisms 475.2.1 Binding of Agonist to Receptor 485.2.1.1 Bonds 485.3 Binding Theory 495.3.1 Early Approaches to Understanding

Receptor Action 495.3.1.1 The Occupancy Model 495.3.1.2 Processes That Follow Receptor

Activation 525.3.1.3 Efficacy and Spare Receptors 525.3.2 Modern Approaches to Receptor

Theory 525.3.2.1 The Two-State Model 52

5.3.2.2 The Ternary Complex Model 535.3.2.3 Protean Agonism 545.3.2.4 Cubic Ternary Complex (CTC)

Model 555.3.3 Summary of Model States 555.4 Visualizing Receptor Structure and

Function 555.4.1 Determination of Receptor Kd 555.4.1.1 Schild Analysis 565.4.2 Visualizing Ligand Binding 575.4.2.1 Receptor Preparation 585.4.2.2 Equilibrium Binding Studies 585.4.2.3 Competition Studies 585.4.3 X-ray Crystallography of Native and

Agonist-Bound Receptors 595.4.4 Probe Tagging (Fluorescent and

Photoaffinity) 605.5 Proteomics Approaches to Receptor

Efficacy 605.6 Physical Factors Affecting Receptor

Binding 615.6.1 Temperature 615.6.2 Relation of Agonist Affinity and Efficacy

to Distance Traveled FollowingRelease 61

Part III Receptor Types andFunction 63

6 Transduction I: Ion Channels andTransporters 65

6.1 Introduction 656.1.1 Family Relationships 656.2 Small Molecule Channels 666.2.1 Osmotic and Stretch Detectors 666.2.2 Voltage-Gated Cation Channels 666.2.2.1 History of Studies on Voltage-Gated

Channels 666.2.2.2 Structure and Physiology of Ion

Channels 686.2.3 Potassium Channels 686.2.4 Sodium Channels 706.2.4.1 Bacterial Na+ Channels 706.2.4.2 Vertebrate Na+ Channels 706.2.5 Calcium Channels 716.2.6 Non-Voltage-Gated Cation

Channels – Transient Receptor Potential(TRP) Channels 72

6.3 Transporters 736.3.1 Pumps and Facilitated Diffusion 736.3.1.1 The SLC Proteins 736.3.1.2 The Pumps 746.3.2 The Chloride Channel 76

Contents IX

6.4 Major Intrinsic Proteins 766.4.1 Water Channels 766.4.2 Glycerol Transporters 776.5 Ligand-Gated Ion Channels 776.5.1 Four-TM Domains – the Cys-Loop

Receptors 776.5.1.1 The Four-TM Channels for Cations 786.5.1.2 The Four-TM Channels for Anions 806.5.2 Three-TM Domains – Ionotropic

Glutamate Receptors 826.5.2.1 Glutamate-Gated Channels 826.5.2.2 N-Methyl-D-aspartate (NMDA)

Receptor 826.5.2.3 Non-NMDA Receptors 826.5.3 Two-TM Domains – ATP-Gated

Receptors (P2X) 82

7 Transduction II: G-Protein-CoupledReceptors 85

7.1 Introduction 857.1.1 Receptor Function 867.1.2 Sensory Transduction 877.1.2.1 Chemoreception in Non-Mammals 877.1.2.2 Chemoreception in Mammals 877.2 Families of G-Protein-Coupled

Receptors 897.3 Transduction Mechanisms 897.3.1 Discovery of Receptor Control of

Metabolism – Cyclic AMP and GProteins 89

7.3.1.1 Components of the Process of MetabolicActivation 89

7.3.1.2 Discovery of Cyclic AMP 907.3.1.3 Discovery of G Proteins 907.3.2 Actions of G Proteins 917.3.2.1 G-Alpha Proteins 927.3.2.2 Roles of the Beta and Gamma

Subunits 957.3.3 Proteins That Enhance (GEF) or Inhibit

(GAP) GTP Binding 967.3.3.1 GEF Protein 967.3.3.2 GAP Protein 967.3.4 Signal Amplification 977.3.5 Signal Cessation – Several Processes

Decrease Receptor Activity 977.3.6 Interactions between Receptors and G

Proteins 977.3.7 Summary of Actions of GPCRs: Agonists,

Receptors, G Proteins, and SignalingCascades 98

7.4 The Major Families of G Protein-CoupledReceptors 99

7.4.1 Family A – Rhodopsin-Like 99

7.4.1.1 The α Subfamily 997.4.1.2 The β Subfamily 1027.4.1.3 The γ Subfamily 1027.4.1.4 The δ Subfamily 1047.4.2 Family B – Secretin-Like 1047.4.3 Family C – Metabotropic Glutamate and

Sweet/Umami Taste Receptors 1047.4.3.1 Taste 1 Receptors (T1Rs) 1057.4.3.2 Calcium-Sensing Receptors 1067.4.4 Family D – Adhesion Receptors 1067.4.5 Family F – Frizzled-Smoothened

Receptors 1067.4.6 Family E – Cyclic AMP Receptors 1067.4.7 Other G-Protein-Coupled Receptor

Types in Eukaryotes 1067.4.7.1 Yeast Mating Pheromone

Receptors 1067.4.7.2 Insect Taste Receptors 1067.4.7.3 Nematode Chemoreceptors 106

8 Transduction III: Receptor Kinases andImmunoglobulins 107

8.1 Protein Kinases 1078.2 Receptors for Cell Division and

Metabolism 1088.2.1 Overview of Family Members 1088.2.2 Overall Functions of RTK 1088.2.2.1 Extracellular Domains 1088.2.2.2 Intracellular Domains 1098.2.3 Receptor Tyrosine Kinase

Subfamilies 1108.2.3.1 EGF Receptor Subfamily 1118.2.3.2 Insulin Receptor Subfamily 1118.2.3.3 FGF and PDGF Receptor

Subfamilies 1118.2.3.4 NGF Receptor Subfamily 1118.3 Receptor Serine/Threonine Kinases 1128.3.1 Transforming Growth Factor-Beta

(TGF-β) Receptor 1128.4 The Guanylyl Cyclase Receptor

Subfamily – Natriuretic PeptideReceptors 112

8.5 Non-Kinase Molecules – LDLReceptors 113

8.5.1 Cholesterol Transport 1138.5.2 The Low-Density Lipoprotein (LDL)

Receptor 1148.5.2.1 Clathrin-Coated Pits 1148.6 Cell–Cell Contact Signaling 1158.6.1 Notch–Delta Signaling 1158.7 Immune System Receptors, Antibodies,

and Cytokines 1158.7.1 The Innate Immune Responses 115

X Contents

8.7.2 The Cells and Molecules of the AdaptiveImmune System 116

8.7.3 T-Cell Receptors andImmunoglobulins 116

8.7.4 Cell-Surface Molecules 1178.7.4.1 The MHC Proteins 1178.7.4.2 Receptors of the B and T Cells 118

9 Transduction IV: Nuclear Receptors 1219.1 Introduction 1219.2 Genomic Actions of Nuclear

Receptors 1229.2.1 Families of Nuclear Receptors 1229.2.2 Transcription Control 1229.2.3 Constitutively Active Nuclear

Receptors 1229.2.4 Liganded Receptors 1229.2.5 History of Steroid Receptor Studies 1239.2.6 Receptor Structure 1239.2.7 The Ligand-Binding Module 1249.2.8 The DNA-Binding Module 1259.2.9 Specific Nuclear Actions 1259.2.9.1 Family 1 – Thyroid Hormone and

Vitamins A and D Receptors 1259.2.9.2 Family 2 – Fatty Acid (HNF4) and

Retinoic X Receptors (RXR) 1279.2.9.3 Family 3 – Steroid Receptors for

Estrogens, Androgens, Progestogens,Mineralocorticoids, andGlucocorticoids 128

9.3 Actions of Receptor Antagonists 1299.4 Non-Traditional Actions of Steroid-Like

Hormones and Their Receptors 1309.4.1 Cell-Membrane Progesterone

Receptors 1319.4.2 Cell-Membrane Mineralocorticoid and

Glucocorticoid Receptors 1319.4.3 Cell-Membrane Thyroid Hormone and

Vitamin A/D Receptors 1319.4.4 Ligand-Independent Activation of

Transcription 131

Part IV Applications 133

10 Signaling Complexity 13510.1 Introduction 13510.2 Experimental Determination of Signaling

Cascades 13510.2.1 Glycolysis 13510.2.2 MAPK: a Phosphorylation Cascade 13610.3 Transduction across the Membrane 13810.3.1 Ion Channels 13810.3.2 G-Protein-Coupled Receptors 138

10.3.2.1 Other G-Protein-LikeTransducers – Ras 139

10.3.2.2 Other G-Protein-LikeTransducers – Ran 139

10.3.3 Cell Aggregation and Development 14010.3.3.1 Coaggregation in Bacteria 14010.3.3.2 Aggregation in Eukaryotes 14010.3.3.3 The Molecules of Cell Adhesion 14110.4 Complexity in Cross Talk – Roles of

PIP3, Akt, and PDK1 14110.4.1 Signaling Cascades Using PIP3 14210.4.2 Integrins 14410.4.3 Receptor Tyrosine Kinases 14410.4.4 Cytokine Receptors and the JAK/STAT

Proteins 14410.4.5 Combined Cellular Signaling – GPCR

and RTK Actions 14410.5 Role in Cancer 14410.5.1 Constitutive versus Inducible

Activation 14410.5.2 Cancer Pathways 14610.6 Signaling Mediated by Gas

Molecules 14610.6.1 Carbon Monoxide 14710.6.2 Nitric Oxide 14710.6.3 Hydrogen Sulfide 148

11 Cellular Interactions inDevelopment 149

11.1 Introduction 14911.2 The Origins of Multicellularity 15011.2.1 Multicellular Lineages in

Prokaryotes 15011.2.2 Multicellular Lineages in

Eukaryotes 15011.2.2.1 Chromalveolates – Generally Unicellular

but with One Multicellular Clade 15111.2.2.2 Archaeplastida – Algae and Plants 15111.2.2.3 Amoebozoans, Fungi, Choanoflagellates,

and Animals 15111.3 The Origin of Symmetry and Axes 15211.3.1 The Multicellular Body Plan 15211.3.2 The Porifera – Asymmetric with a Single

Cell Layer 15211.3.3 Cnidaria – Radial Symmetry, Two Cell

Layers, Tissues 15311.3.4 Mesoderm 15411.4 Fertilization and Organization of the

Multicellular Body Plan 15411.4.1 Sperm–Egg Recognition 15411.4.1.1 Sea Urchin Fertilization 15411.4.1.2 Mammalian Fertilization 157

Contents XI

11.5 Differentiation of TriploblasticEmbryos – Organogenesis 158

11.5.1 Introduction 15811.5.2 The Origin of Triploblastic Animals 15811.5.3 Development in Protostomes 15911.5.3.1 Segmentation and Organ Formation in

Drosophila 15911.5.3.2 Cellular Interactions in Later Drosophila

Development 16111.5.4 Development in Deuterostomes 16211.5.4.1 Early Frog Development 16211.5.4.2 Nerve Growth 16411.6 Programmed Cell Death

(Apoptosis) 16511.6.1 Apoptosis During Development 16611.6.2 Apoptosis During Adult Life 166

12 Receptor Mechanisms in DiseaseProcesses 169

12.1 Genetic Basis for ReceptorFunction 169

12.1.1 Genotype and Phenotype 16912.1.2 Classical Dominance Mechanisms 16912.1.3 Other Levels of Gene Expression 17012.1.4 Pre-receptor Mutations 17012.1.5 Receptor Mutations 17112.1.6 Post-receptor Mutations 17112.2 Receptor Pathologies 17112.2.1 Ion Channel Superfamily 17112.2.1.1 Calcium Channels 17212.2.1.2 Transient Receptor Protein (TRP)

Channels 17212.2.1.3 Voltage-Gated Na+ Channels 17212.2.1.4 Ligand-Gated Na+ Channels 17212.2.1.5 Chloride Transporter – Cystic

Fibrosis 17212.2.2 G-Protein-Coupled Receptor

Superfamily 17212.2.2.1 Cholera 17212.2.2.2 Thyroid Diseases 17312.2.2.3 Cardiovascular Disease 17312.2.2.4 Obesity 17412.2.2.5 Depression 17512.2.2.6 Schizophrenia 17512.2.3 Immunoglobulin Superfamily 17612.2.3.1 Diabetes Mellitus 17612.2.3.2 Atherosclerosis 17612.2.4 Nuclear Receptor Superfamily – Steroid

Receptors 17612.2.4.1 Alterations in Transcription 17612.2.4.2 Additional Effects 17712.3 Signaling Mutations Leading to

Cancer 177

12.3.1 Pathogenesis of Cancer 17712.3.2 Cancer as a Disease of Signaling

Molecules 17812.3.2.1 Oncogenes that Encode Mutated

Transmitters 17812.3.2.2 Oncogenes that Encode Mutated

RTKs 17812.3.2.3 Oncogenes that Encode Mutated G

Proteins 17912.3.2.4 Oncogenes that Encode Mutated

Transcription Factors – SteroidReceptors 180

13 Receptors and the Mind 18113.1 Origins of Behavior 18113.1.1 Bacterial Short-Term Memory 18113.1.2 Animals Without True Neural

Organization: The Porifera 18213.1.3 Animals with Neural Networks: The

Cnidaria 18213.1.4 Bilaterally Symmetrical Animals: The

Acoela 18313.2 Nervous Systems 18313.2.1 Organization 18313.2.2 Neurons 18313.2.2.1 Cell Structure 18313.2.2.2 Mechanisms 18413.2.3 Transmitters 18413.2.3.1 Synthesis and Release of Brain

Transmitters 18513.2.3.2 Converting Short-Term Memory to Long

Term 18613.3 Animal Memory: Invertebrates 18613.3.1 Discovery of the Signaling Contribution

to Memory 18613.3.2 Receptor Mechanisms of Nerve Cell

Interactions 18613.3.2.1 The Gill Withdrawal Reflex of

Aplysia 18613.3.2.2 Mechanisms Underlying Sensitization

and Short-Term Memory 18713.3.2.3 Ion Flows in Nerve Action

Potentials 18713.3.2.4 Consolidation into Long-Term Memory

(LTP) 18813.4 Animal Memory: Vertebrates 18813.4.1 Intracellular Mechanisms of

Potentiation 18813.5 Receptors and Behavior: Addiction,

Tolerance, and Dependence 19013.5.1 Opioid Receptors 19013.5.1.1 Opioid Neuron Pathways in the

Brain 191

XII Contents

13.5.1.2 The Opioid Peptides and Receptors 19213.5.1.3 Mechanisms of Transduction 19213.5.1.4 Characteristics of Responses to

Continued Drug Presence 19213.5.2 Individual and Cultural Distributions of

Depression 19313.5.2.1 Depression 19313.5.2.2 Polymorphisms in Neurotransmitter

Transporters 19413.5.2.3 Polymorphisms in Opioid Receptor

Subtypes 19413.5.2.4 Polymorphisms in Enzymes for

Transmitter Disposition 19413.5.2.5 Society-Level Actions 19413.5.2.6 Possible Mechanisms 195

14 Evolution of Receptors, Transmitters, andHormones 197

14.1 Introduction 19714.1.1 Phylogeny of Communication: General

Ideas 19714.1.2 The Receptors 19714.2 Origins of Transmitters and

Receptors 19714.2.1 Evolution of Signaling Processes 19714.2.2 Homologous Sequences 19814.2.2.1 Orthologous and Paralogous

Sequences 19814.2.3 Phylogenetic Inference 19914.2.4 Phylogenetic Illustration of Protein

Relationships 19914.2.5 Whole-Genome Duplication

(WGD) 20014.2.6 Origins of Novel Domains 20114.2.7 Adaptation of Receptor Systems 20114.2.8 Coevolution of Components of Signaling

Pathways 20214.2.9 Peptide Hormones and Their

Receptors 20214.2.10 Receptors and Their Non-Peptide

Hormones 20214.3 Evolution of Hormones 20214.3.1 Peptide Hormones for G

Protein-Coupled Receptors 20214.3.1.1 The Yeast Mating Pheromones 203

14.3.1.2 The Anterior Pituitary TrophicHormones 203

14.3.1.3 The Hypothalamic ReleasingHormones 203

14.3.1.4 The Posterior Pituitary Hormones 20314.3.1.5 Miscellaneous Peptide Hormones 20414.3.2 Hormones of the Receptor Tyrosine

Kinases 20414.3.2.1 The Insulin Family 20414.3.2.2 The Neurotrophins 20414.3.2.3 The Growth Hormone Family 20414.4 Evolution of Receptor

Superfamilies 20514.4.1 Ion Channels 20514.4.1.1 Voltage-Gated Channels 20514.4.1.2 Ligand-Gated Channels 20514.4.2 G Protein-Coupled Receptors 20614.4.2.1 G-Protein-Coupled Receptor Types 20614.4.2.2 Family A Receptors – Rhodopsin

Family 20614.4.2.3 Family B – Secretin and Adhesion

Receptors 20714.4.2.4 Family F – Frizzled and Smoothened

Receptors 20814.4.2.5 Elements of the GPCR Transduction

Pathway 20814.4.3 The Immunoglobulin Superfamily 21014.4.3.1 The Receptor Tyrosine Kinases 21014.4.3.2 Molecules of the Adaptive Immune

System 21114.4.4 Steroid, Vitamin A/D, and Thyroid

Hormone Receptors 21114.4.4.1 Origin of Nuclear Receptors: The Role of

Ligands 21114.4.4.2 The Nuclear Receptor Families 21114.4.4.3 Later Evolution of Nuclear

Receptors – Ligand Exploitation 21214.5 Evolution of Receptor Antagonism 21314.6 A Final Note 213

Glossary 215

References 227

Index 241

XIII

Acknowledgments

Drs Kent Thornburg and George Olsen (OregonHealth and Sciences University) devoted much timeand thought to an early version of the manuscript,and made valuable comments at all levels. Dr PaulKolenbrander (National Institutes of Health) pro-vided valuable insights regarding bacterial signaling.Drs Christian Burvenich and Eddy Roets (Universityof Ghent) and Erik Raman (University of Antwerp)were valued colleagues and mentors in receptorpharmacology during MR’s sabbatical research inBelgium. Numerous colleagues at the Mayo Clinicwere helpful mentors in cancer biology and receptorsignaling during AK’s postdoctoral fellowship.

We also thank our Linfield students and col-leagues as sources of assistance and stimulation. JohnSyring gave a valuable critical reading of the chapteron receptor evolution. Linfield students ChelseyNieman, John Frank, Bonnie Hastings, Eric Lemieux,Chelan Guischer, Jacob Priester, Christine Lewis,and Henry Simons gave valuable suggestions and

editorial assistance. Lige Armstrong of the LinfieldLibrary Faculty Development Laboratory, providedassistance with illustrations. In addition, Dr MirandaByse (Linfield graduate) read parts of the manuscriptand worked with MR on signaling experiments.

We are also pleased to acknowledge the scientificand editorial assistance of the editors at Wiley-Blackwell, especially Dr Gregor Cicchetti, Ms AnneDuGuerny, and Ms Stefanie Volk.

Finally, other members of the Biology Departmentat Linfield College made the thinking and writing pro-cess especially enjoyable, and we thank them for theircollegiality and conversations concerning the book.

“Drinking coffee with people cleverer than oneself isnot a waste of time, but one of the best ways of

expanding horizons.”David Colquhoun, 2006

Part IIntroduction

3

1Introduction

The beauty of reductionism is that it gives yousomething to do next.

Steve Jones [1]

Biological processes require communication betweencells and between individuals. In all kinds of livingorganisms, this communication begins at the molec-ular level. Small signaling molecules (proteins,amino acids, steroids, and other substances) are themessages that pass from one cell to the next; largeprotein receptors are the receivers of the message.Receptors bind the smaller molecules much as alock receives a key or a glove receives a hand [2].Other proteins in the cell membrane associated withthe receptors convey the message to the interior ofthe cell.

Very few biochemical or physiological functionsin our bodies are not somehow touched by thesemolecules or by the process of cellular communica-tion. Here are some examples of how receptors areinvolved in a variety of biological processes:

• Sperm and egg meet, recognize each other, and bindby a receptor mechanism.

• Embryos develop by cell communication: onecell releases a hormone that binds to a receptoron another cell, and the second cell changesits shape and function, initiating the process ofdifferentiation.

• Hormone-like neurotransmitters are releasedfrom one cell (a nerve) and bind to receptors on thesurface of a nearby cell (another nerve or a muscle)to cause thought or movement.

• The digestive system propels food and releasesenzymes according to the binding of hormones tocells lining the digestive tract.

• Immune system cells contain on their surfacesreceptors that are able to recognize foreignproteins and attack invading cells.

• Diseases often act by subverting normal receptorfunction.

This introductory chapter covers general conceptsof communication and how chemical communicationcompares with human communication; how evolu-tion applies to receptor molecules; and how a purechemical entity such as a receptor can initiate suchlarge-scale functions as thought.

1.1Receptors and Signaling

1.1.1General Aspects of Signaling

Signaling is the means by which a cell knows what ishappening in its surroundings, and is also the methodby which one cell instructs nearby cells to alter theirbehavior. Organismal cell signaling involves molecu-lar interactions, but the biological mechanisms of sig-naling are analogous to the ones humans use for verbalcommunication.

1.1.2Verbal and Physiological Signals

Any sort of signaling requires that the sender andreceiver are capable of interpreting the signals in thesame way [3]:

• The sender must relay a characteristic signal, and itmust be received by a characteristic device;

• The signal is arbitrary: it bears no real relation tothe process it starts but is simply a way of obtaininga response in the receiver;

• The signal is simpler than the process it sets inmotion.

These rules are easily understood in terms of humancommunication:

• The signals are the words of the language, and thereceiver is the hearing/thinking/acting apparatus ofanother person;

Receptor Biology, First Edition. Michael F. Roberts and Anne E. Kruchten.© 2016 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2016 by Wiley-VCH Verlag GmbH & Co. KGaA.

4 1 Introduction

• Each language uses different words, yet all peoplecan express the same thoughts.

• Any word (e.g., HELP) evokes in its hearer a set ofthoughts or behaviors that are much more complexthan the word itself.

The units of cellular communication abide by thesesame rules:

• The correct signal is the drug or hormone, the cor-rect receiver is the cell surface receptor or nuclearreceptor.

• It is arbitrary that one amino acid (e.g., glutamicacid) is an excitatory transmitter in the nervoussystem, whereas another amino acid (e.g., glycine)is an inhibitory transmitter.

• The binding of a single transmitter molecule to itsreceptor is adequate to start a cascade of intracel-lular events that amplifies the signal into a complexbiochemical response.

In addition to these constraints, three more gener-ally apply to biological communication:

• The receptor must be present on the correct tissue,it must be selective or specific to the hormone, andthe receptor must not be present in tissues wherethe response is not desired; thus, the timing of themessage must be coordinated with the presence ofthe receptor for that message.

• The signal must always mean the same thing to aparticular receptor–effector mechanism.

• Some transmitters act on more than one typeof receptor, often activating antagonistic cellularprocesses.

The analogies drawn between human communica-tion and chemical communication are symbolic, yetthe correspondence between the two systems is beingstrengthened as we find more instances where humaninteractions are being found to be at least partlychemical (e.g., the importance of pheromone-likesubstances in human behavior [4]).

1.1.3Criteria for Recognizing Transmitters and Receptors

This book refers to signaling molecules in severalways. The most general term is ligand, which meansany molecule that binds to a receptor. A ligandthat activates its receptor is called an agonist.Hormones, transmitters, and pheromones are allagonists, and are naturally produced by organisms forsignaling.

1.1.4Agonists

The substances that serve as agonists are often alsoimportant as metabolic molecules within the cell.Thus, simply showing that a cell produces acetyl-choline, for example, does not demonstrate its roleas a transmitter. For a substance to be acceptedas a specific transmitter or hormone, it must beshown to: [5]

• be synthesized, stored, and released from theproper type of cell (e.g., neuron or endocrine cell);

• have a specific mechanism for removal from theextracellular space near the target cell;

• be effective as an agonist if added to the target cellby experimenters.

1.1.5Receptors

Cells can be activated by processes other than recep-tor mechanisms. To be accepted as a receptor mecha-nism, a process must be shown to [6]

• be activated by one or only a few substances;• bind these substances with high affinity;• be able to transmit the binding event to the cell

interior.

These criteria for identifying receptors are notjust for convenience; each has its basis in receptorstructure, and later chapters show how these criteriaare derived from, and actually define, the molecularmechanisms by which receptors operate.

1.1.6Receptor–Enzyme Similarities

Enzymes are familiar proteins: they have active sitesat which small substrate molecules bind and are con-verted to products. The relation between a receptorand its agonist is quite similar, at least at the bind-ing step, to the action of enzymes: the receptor bindsthe agonist with high affinity because of the matchbetween the shape and electric charge distribution ofboth molecules. The act of binding alters the shape ofthe receptor at another location; this change in shapeis transmitted to other cellular proteins, thus stimu-lating further cellular processes.

As useful as the enzyme analogy is, however,enzyme action is unlike the receptor mechanism insome ways:

1.2 Types of Receptors and Hormones 5

Table 1.1 Locations and properties of the four receptor superfamilies.

Ion channel receptors G-protein-coupled receptors Receptor tyrosine kinases Nuclear receptors

Location Plasma membrane Plasma membrane Plasma membrane NucleusEffector Ion channel Enzyme or ion channel Enzyme Regulation of gene actionTime scale Milliseconds–seconds Seconds–minutes Minutes–hours Hours–daysExamples Nicotinic receptors Adrenoceptors Insulin receptors Steroid receptors

• A receptor-binding event has no “product” becausethe agonist is unaltered by its interaction with thereceptor.

• The receptor–agonist complex has an additionalrole after binding: the conversion of the bindingsignal to an intracellular event, such as enzymeactivation or gene transcription.

Enzymes are important intracellular biochemicalregulators; receptors are important regulators at theinterface of the cell. Because of this location, theyhave a crucial role as molecular guardians, controllingthe initial encounters between cells and chemicals intheir environments.

1.2Types of Receptors and Hormones

1.2.1Receptor Superfamilies

A protein superfamily is a group of proteins thatshare structure, sequence, and functional featuressuggesting they are derived from the same commonancestral protein [5]. At present, researchers rec-ognize four large superfamilies1 of receptors: threereside in the cell membrane and one remains withinthe cytoplasm of the cell. Almost a thousand types ofcell surface receptors belong to a single superfamily,the G-protein-coupled receptors. Their DNA thuscomprises about 5% of all human genes. Another largesuperfamily of receptors is the fast ion channels thatmediate neurotransmission in the central nervoussystem and skeletal muscles. A small superfamily, thereceptor kinases, mediates metabolic, developmental,

1 Several terms are used in receptor literature to denote classes ofreceptors: superfamily, family, class, group, and clan, often incon-sistently. We use the term superfamily to refer only to the fourmajor groups of receptors, and use the other terms in order: withineach superfamily are found families (e.g., the several types of ionchannels); within each family are found classes (e.g., the types ofCa2+ channel). Group will be used informally and sparingly, clannot at all.

and immunological processes (Chapter 8). Alsopresent in small quantities in the cytoplasm of the cellare the nuclear receptors that control transcription ofnew proteins. Table 1.1 summarizes the properties ofthe four superfamilies or receptors [7].

These four types are easily distinguished by shape,they each have characteristic agonists, and eachcauses characteristic intracellular changes. Figure 1.1shows general structures of the four superfamilies.

The first superfamily (Chapter 6) consists of ionchannels such as the nerve Na+ channel that isactivated by acetylcholine. These receptors consist ofseveral protein chains held together in a ring. Eachprotein has four transmembrane regions. Togetherthe separate chains form the pore through which theNa+ ion moves when the agonist binds. The inwardflux of Na+ depolarizes the cell, causing it to generatean action potential.

The second superfamily (Chapter 7) consists ofreceptors such as the one for the neurotransmitternorepinephrine (NE) on heart muscle cells. Thisreceptor has seven transmembrane regions, meaningthe single receptor molecule passes through the cellmembrane seven times and has both intracellularand extracellular regions. When a transmitter suchas NE binds, it causes the receptor to activate a mul-tiprotein assemblage in the membrane that producesan intracellular second messenger (such as cyclicadenosine monophosphate (cAMP)) that activatesthe cell by altering the level of phosphorylation ofcellular enzymes. In the heart muscle, one effect ofNE is to increase the strength of the heartbeat.

The third superfamily (Chapter 8) consists of thereceptor kinases, growth factor receptors for sub-stances such as the proteins insulin and epidermalgrowth factor. These receptors have a single trans-membrane region, and their cytoplasmic end is anenzyme – a kinase. The binding of the hormone to theouter portion activates the kinase to phosphorylatecellular enzymes that regulate nutrient transport andcell division.

The fourth superfamily (Chapter 9) consists ofthe intracellular receptors, the proteins that bind

6 1 Introduction

Out

In

Ionchannel

G-protein-coupledreceptor Receptor

tyrosinekinase

Cellmembrane

Nuclearreceptor

Figure 1.1 Overview of the four major receptor types. (A) Ion channel with extracellular domain labeled in red and transmembranechains labeled in green; (B) G-protein-coupled receptor with extracellular domains labeled in red, seven transmembrane domainslabeled in green; (C) receptor tyrosine kinase with transmembrane domain labeled in green, extracellular regions in red, and intracel-lular regions in blue. Black lines represent lipid bilayer; and (D) nucleus (dashed line) with nuclear receptors labeled in blue dimerizingon a DNA template labeled in black. Images were created using Rasmol [8] from PDB ID [9], PDB ID 1F88 [10], PDB ID [11], and PDBID [12].

steroids, thyroid hormone, and certain vitamins.These fat-soluble ligands diffuse through the cellmembrane to the interior of the cell, where theybind to and activate receptor proteins that enhancesynthesis of new proteins within the cell.

Receptors are involved in cellular processes, such asmetabolism and ionic changes, as well as cell division,growth, and protein synthesis. This book also coversreceptor actions at a higher, organismal level. Embry-onic development (Chapter 11), disease (Chapter12), and the activities of the mind (Chapter 13) allinvolve integration of many physiological systems,all bound by the same receptor process as cell–cellcommunication.

All classes of receptors are encoded by geneswithin each cell. Genes for receptors are also subjectto mutation and evolve by natural selection. Asa consequence, receptors will change over time,allowing us to draw evolutionary inferences fromthe present phylogenetic distribution of genes forfamilies of receptor molecules. The “fossil record”of proteins is thus found not in the rocks of theworld but in the diversity of present-day organisms.The four superfamilies of receptors described are allwidespread in eukaryotes. Some superfamilies arealso present in prokaryotes, and the study of theirdistribution among all organisms (Chapter 14) gives

researchers an understanding of their functions androle in organismal adaptations.

The relationships among protein families suggestthat their genes have mutated, changed location,and duplicated many times, each time allowing theproduction of new protein molecules with similarfunctions. These similarities indicate further thatprotein function can change over time, and that newproteins with completely different functions can arisefrom gene mutations. This seems to be how somereceptors arose, and how the families of receptorshave changed.

1.3Receptors Are the Chemical Expression of Reality

Because receptors are at the interface between cellsand their environments, they are the first cellularunits to receive environmental information andprovide crucial information about the surroundings.For example, animals know that nighttime is thetime to sleep, even though their brains have noway of directly sensing the light or dark. Visualinformation from the eyes goes to the pineal gland,which produces the hormone melatonin in inverseproportion to the amount of ambient light. Melatoninis therefore the chemical expression of darkness [13].

1.3 Receptors Are the Chemical Expression of Reality 7

In an analogous manner, other hormone and receptorsystems give information about the food taken inby organisms. Insulin is produced in the pancreasfollowing a meal when blood glucose levels rise.Insulin is therefore the chemical expression of plenty.When food is scarce, the adrenal gland producesthe steroid cortisol as a means of liberating glucosefrom storage forms in cells. Cortisol can be seen asthe chemical expression of starvation. As melatonin,insulin, and cortisol all act on cellular receptors, weview receptor mechanisms as an important way thatorganisms have of knowing what reality is.

As the foregoing suggests, receptors are complex,as are their interactions with cellular processes.However, we hope that this complexity will be madecomprehensible by the approach we are taking:the thousands of different receptors fall into onlyfour fundamental superfamilies; each has a uniquestructure and a unique way of activating the cell,so it is possible to identify an unfamiliar receptor ifone knows only a few things about it. Knowledge ofreceptor function illuminates the many interactionsamong proteins in the body and gives researchersimportant information on higher level functionsof cell physiology (e.g., the normal workings of themind or the aberrant interactions involved in diseasestates).

The book is divided into three parts: first is ageneral discussion of cell membranes, proteins, hor-mone types, and receptor theory.2 Next follows onechapter on each of the four receptor types. Finally,several chapters outline receptor-mediated biologicalprocesses such as embryonic development, disease,mechanisms of the mind, and the evolution of theseremarkable molecules.

Pharmacology texts generally focus on hormonesand the kinetics of drug actions. We have writtenthis book for students who wish to become morefamiliar with receptors themselves: the mechanismsby which they act, the sorts of processes they direct,and their evolution as molecules. It is meant forstudents at the advanced undergraduate and early

2 The term theory is often used by mistake in place of hypothesis. Inproper usage, a theory is a hypothesis that has been tested and pro-moted to the level of widespread acceptance as a major concept inscience. It is unfortunate that scientists themselves often misusetheory to mean hypothesis, as in “I have a theory about that” andnon-scientists often pounce on this misuse to denigrate science,as in “evolution is only a theory.” In this book, we restrict the useof the term theory to major scientific concepts, such as the theoryof evolution, or cell theory, or receptor theory. All three of theseideas have been rigorously tested; they are no longer hypotheti-cal, but have become key concepts in biological thinking. Otherconcepts, still provisional, are called hypotheses.

graduate levels and requires an understanding offundamental chemical and biological principles, ageneral knowledge of evolutionary thought, and agrasp of physiological interactions – all concepts thatare part of any good general biology course. The textbuilds on these ideas to help students form a morecomplex understanding of pharmacology and cellularbiochemistry.

Evolutionary inferences provide information thatallows the study of receptors to be not only excitingand useful but also conceptually possible: despite thebewildering array of cell surface receptor types, theyfall into just four major categories and interact withonly a few dozen other membrane effector proteinsthat transmit the binding event into a biochemicalprocess. Thus, genetic relationships among recep-tors are relatively simple, and their use of similarbiochemical mechanisms shows that the importantproblems of cell-to-cell signaling have needed to besolved only a few times in evolution.

We wrote the book because in our teaching andresearch we see the importance of receptor mecha-nisms and intracellular signaling across all kingdomsof organisms and in many types of cellular processes.Even so, it is difficult to find a book that gives themcomplete coverage (structure, mechanism of action,evolutionary history) without being written specif-ically for professionals. The two unifying themes ofthe book are (i) the receptor concept itself: the ideathat biological communication is involved in nearlyall the activities of living things, and that receptorfunction is the mechanism of that communicationand (ii) the role of natural selection and evolution inshaping receptor structure and function. We hopethat this book will give a clear idea of the roles thathormones and their receptors play in our lives, fromthe reactions of individual cells to the behavior ofwhole organisms.

9

2The Origins of Chemical Thinking

A mystery is a phenomenon that people don’t knowhow to think about – yet.

Daniel Dennett [14]

2.1Overview of Early Pharmacological History

2.1.1The Development of a Chemical Hypothesis

The earliest Greek thinkers such as Thales (sixthcentury BCE) and Democritus (fourth century BCE)taught that life was material and that physical com-ponents of the environment were responsible for theorganization of matter into living things. Thales alsoinitiated an experimental approach to studying nat-ural phenomena [15]. However, these early thinkerswere unusual – the scholars who followed them hada non-material, non-experimental, non-molecularconcept of the world. The non-material worldviewpromoted the idea that life processes were fundamen-tally different from processes in non-living systems.The non-experimental worldview encouraged theuse of logic rather than the use of experiment to testideas about natural phenomena. The non-molecularworldview is best seen by its two main hypothesesconcerning the physical and biological spheres: thefour “elements” (earth, air, fire, and water) and the“humoral” hypothesis (yellow bile, black bile, blood,and phlegm).

Plato (fourth century BCE) exemplified the non-material view, as he deemphasized observation andexperiment, and claimed that our perceptions ofmatter were transitory and only what the mindperceived (via logic) was permanent [15]. Underhis system, mind and body were considered to beseparate entities – the senses give an inaccurateversion of the world; only the mind provides “purity”of perception [16]. Aristotle’s (fourth century BCE)and Galen’s (second century CE) thinking opposed

this attitude, as they appreciated the role of matterin life and advocated experimental approaches to thestudy of nature.

Descartes (seventeenth century CE), although aproponent of reason and experimentation, main-tained that the body is a dual being, both mind andmatter, and the workings of the mind are outsidenature. He said that because mechanism describesnon-human workings, then other laws must apply tohuman workings [17]. The concept of mind–bodydualism of Descartes and others thus furtherednon-material approaches to the study of life, andinhibited development of a systematic approach tothe study of, among other things, the function ofthe brain.

However, chemical thinking did arise among somemedieval thinkers: the physician Paracelsus in thesixteenth century was the first to take up the con-cepts of the earliest Greeks, teaching that the bodywas composed of chemicals and that illnesses werethe result of chemical imbalances. He anticipatedmodern thinking in two of his teachings: that withina natural product the curative agent could be asingle substance, and that the curative or poisonousfunctions of a drug were directly proportional to itsconcentration [18].

Felix Fontana in the late eighteenth century experi-mentally confirmed Paracelsus’ view that a crude drugexerted its effect through a specific active principlethat acted on a discrete tissue in the organism. Hiscontemporary Peter Daries showed further that theeffect was proportional to the concentration of drugapplied. Setürner in the early nineteenth centurywas the first actually to isolate a pure drug when heobtained morphine from opium. This achievementinitiated a period of rapid change: before manydecades had passed, the chemical natures of manypharmacological substances were determined, andthe new drug manuals, or pharmacopoeias, werebased on pure substances rather than on crudeplant extracts. Organic chemistry was emerging as

Receptor Biology, First Edition. Michael F. Roberts and Anne E. Kruchten.© 2016 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2016 by Wiley-VCH Verlag GmbH & Co. KGaA.

10 2 The Origins of Chemical Thinking

a discipline in its own right, and the elucidationof chemical structure soon led to the develop-ment of drugs from synthetic sources rather thannatural ones.

2.1.2Chemical Structure and Drug Action

The three principal researchers in this area in theearly-to-middle nineteenth century were FrançoisMagendie and his students Claude Bernard andJames Blake. Magendie was the author of the firstmodern physiology textbook [19], initiated thestudy of experimental pharmacology, and was thefirst to show that drugs act on specific organs.Blake showed that related chemical compounds haddiffering effectiveness in activating tissues. Fromexperiments with chemically related inorganic salts,Blake developed the concept that the biological effectof a substance depended on its structure and chem-ical nature. He found in general that the biologicaleffects of different metallic ions fell into predictablepatterns; these groupings based on biological activityactually predated Mendeleev’s periodic chart of theelements, which was based on inorganic chemicalcombinations [20].

2.1.3The Site of Drug Action

It is only in the last century or so that material,experimental, and chemical principles have beenapplied consistently to help us understand biologicalprocesses. The mechanisms of drug–tissue inter-actions were developed by Bernard [21], who madeimportant discoveries with one particular tissue, thenerve–muscle junction in the skeletal muscle of thefrog. He showed that curare, the South AmericanIndian arrow poison, paralyzes muscles by acting ona structure in the junction (synapse) between thenerve and the muscle. We now know that curarebinds to but does not activate the same receptorto which the neurotransmitter acetylcholine (ACh)binds. Curare thus antagonizes the transmitter actionof ACh (Figure 2.1).

Bernard’s experiments in the 1840s and 1850swere thus the earliest demonstrations of the exis-tence of receptors. He also showed the existenceof the nerve–muscle synapse and established thatnerves and muscles were separate cells. It would be50 years before any of these ideas would be developedtheoretically and experimentally by others.

2.2Modern Pharmacology

2.2.1Langley and Ehrlich: the Origins of the Receptor Concept

John Langley was a student at Cambridge Universityin 1875 when he began studies of the actions of twoalkaloids, atropine and pilocarpine, on the heart.He found that pilocarpine slowed the heart and thatatropine opposed the action of pilocarpine. Further,the way atropine worked suggested to him that itwas acting at the same location as pilocarpine, andhe proposed that both substances had affinity for thesame site, for which both had binding affinity [22].He did not at that time propose the molecular natureof the site, although it is clear that his thinking wasgoing in that direction. Langley’s research interestschanged for a quarter century, and only after 1900did he return to work on the receptor concept [23].

Paul Ehrlich also began his studies of specificbinding processes in the 1870s. His initial work waswith dyes used in histological staining of tissues forpreparation of microscope slides. He proposed thatthe dyes used to stain cells did not simply adherenonspecifically to the material but that they showeda chemical affinity for certain molecules in the cell. Insucceeding years, he continued to lay the frameworkfor the concept of a definite cell-surface molecule thatacted in cell–cell communication [24]. He was bythis time concerned with the genesis of the immuneresponse; he discovered mast cells and proposed thecurrently used classification system for leukocytesbased on cytoplasmic granulation [25]. Ehrlich alsoproposed that immune system cells have the abilityto attract and bind foreign substances with specificchemical affinities to the immune cell surface [26].

Even though Ehrlich had developed the conceptof specific binding for immune system activation,he borrowed the descriptive term “lock and key”from Fischer, who in 1894 had proposed a specificmechanism of binding for enzymes and substratesin biochemical reactions [27]. Even though thelock-in-key interaction is now better thought of as an“induced fit” interaction [2], Ehrlich’s thinking aboutreceptors was modern: he conceived of a receptoras having a special active site with a high affinity forthe foreign chemical. He also anticipated the currentimmunological concept that when an immune systemcell binds an antigen, receptors for the antigen are,as a result, manufactured in large numbers and act inthe blood as binding agents for the antigen [26].

2.2 Modern Pharmacology 11

Synaptic vesicle

Presynapticneuron

Synapticvesicle fusing

Neurotransmitter released

Presynaptic membrane

Postsynaptictransmitterreceptor Ions flow through

postsynaptic channels

No ions flow throughpostsynaptic channels

Synapticcleft

Synapticcleft

Postsynapticmembrane

Curare

(a)

(b)

Postsynapticneuron

Figure 2.1 (a) Axonal ending of a neuron at a postsynaptic cell (nerve or skeletal muscle). The axon is not directly connected to themuscle cell, but is separated by a gap or synapse. The transmitter (green spheres) is synthesized in the ending within vesicles; it isreleased in response to an action potential. The transmitter diffuses to receptors on the postsynaptic cell. (b) An antagonist such ascurare (red ovals) binds to the same receptor at the active site, preventing the transmitter from binding and thereby preventing theeffects of nerve stimulation. http://kin450-neurophysiology.wikispaces.com/Synaptic+Transmission. Accessed 7 February 2015.