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G Protein Coupled Receptors - 1st Edition - ISBN: 9780123918611, 9780123918727

G Protein Coupled Receptors, Volume 520

1st Edition


Serial Volume Editor: P. Michael Conn
Hardcover ISBN: 9780123918611
eBook ISBN: 9780123918727
Imprint: Academic Press
Published Date: 8th February 2013
Page Count: 440
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Table of Contents

Series Page



Methods in Enzymology

Chapter One. Expression of GPCRs in Pichia pastoris for Structural Studies

1 Introduction

2 Generation of Expression Clones

3 Screening Transformants

4 Characterization and Optimization of Initial Target Receptor Production

5 Large-Scale Production

6 Prospects for Large-Scale GPCR Production in P. pastoris


Chapter Two. Conformational Ensemble View of G Protein-Coupled Receptors and the Effect of Mutations and Ligand Binding

1 Introduction

2 Overview of the Conformational Ensemble Prediction

3 Generating Starting Structures Based on Templates

4 BiHelix: TM Bundle Conformational Sampling of Helix Rotation Angles

5 SuperBiHelix TM Bundle Sampling of All Helix Orientation Angles

6 Effect of Mutations on the WT Conformational Ensemble

7 Ensemble Docking of Ligands to WT or Mutant Receptors

8 Summary


Chapter Three. Structural Evolution of G-Protein-Coupled Receptors


1 Introduction

2 The Puzzle of GPCR Evolution

3 The Evolution of Class A GPCRs Viewed by MDS

4 Evolutionary Trends

5 Structural Evolution of GPCRs

6 Summary


Chapter Four. Directed Evolution of G-Protein-Coupled Receptors for High Functional Expression and Detergent Stability

1 Introduction

2 Methods


Chapter Five. The Role of Hydrophobic Amino Acids in the Structure and Function of the Rhodopsin Family of G Protein-Coupled Receptors

1 Introduction

2 The Structure of GPCRs

3 Sequence Analyses of the 7TM Segments of the Rhodopsin Family of GPCRs

4 Importance of the Highly Conserved Hydrophobic Amino Acid at Position 3.40 in the Process of Agonist-Induced Receptor Activation

5 The Hydrophobic Cages of Arginine of the (D/E)RY Motif in TM3, Tyrosine of the (N/D)PxxY Motif in TM7, and Tyrosine in TM5

6 The Transmembrane Aqueous Channel is Interrupted by a Layer of Hydrophobic Residues

7 The Role of Highly Conserved Hydrophobic Residues in G Protein Binding

8 The Role of Highly Conserved Hydrophobic Residues in Receptor Oligomerization

9 Conclusions


Chapter Six. Structure of β-Adrenergic Receptors

1 Introduction

2 Toward the Structures of β-Adrenergic Receptors

3 Lessons from the Structures of β-Adrenergic Receptors

4 Outlook


Chapter Seven. Advances in Methods to Characterize Ligand-Induced Ionic Lock and Rotamer Toggle Molecular Switch in G Protein-Coupled Receptors

1 Introduction

2 Experimental Principles

3 Detailed Experimental Procedure


Chapter Eight. Crystallogenesis of Adenosine A2A Receptor—T4 Lysozyme Fusion Protein

1 Introduction

2 Generation of Fusion Receptors for Crystallogenesis

3 Small-Scale Purification

4 Analytical Size-Exclusion Chromatography

5 Other Analytical Methods

6 Ligand and Additive Selection

7 Large-Scale Purification of Receptors

8 Crystallization

9 Crystal Testing and Data Collection

10 Summary


Chapter Nine. Probing GPCR Structure

1 Introduction

2 Receptors for Extracellular Nucleosides and their Modeled Structures

3 Receptors for Extracellular Nucleotides and their Modeled Structures

4 Neoceptors: Reengineering GPCRs for Recognition of Modified Agonists


Chapter Ten. Strategies for Studying the Ligand Binding Site of GPCRs


1 Introduction

2 The VPAC1 Receptor, an Archetype of the Class B GPCRs

3 Photoaffinity Labeling of the VPAC1 Receptor

4 Chemical and Enzymatic Cleavages of Photoaffinity-Labeled VPAC1

5 Identification of the VPAC1 Residue Covalently Linked to the 125I-Bpa Probe by Edman Degradation Sequencing or “Met-Scan” Procedures

6 Summary


Chapter Eleven. Expression of Mammalian G Protein-Coupled Receptors in Caenorhabditis elegans

1 Introduction

2 Expression of Transgenic GPCRs in C. elegans

3 Detection of Heterologous GPCR Gene Expression

4 Large-Scale Expression of Heterologous GPCRs

5 Purification of Heterologous GPCRs

6 Determination of Transgenic GPCR Activity and Function

7 Concluding Remarks


Chapter Twelve. Expression, Purification, and Structural Analysis of Intracellular C-Termini from Metabotropic Glutamate Receptors


1 Introduction

2 Synthesis of mGluR-CT

3 Structural Analysis


Chapter Thirteen. Unnatural Amino Acid Mutagenesis of GPCRs Using Amber Codon Suppression and Bioorthogonal Labeling

1 Introduction

2 Site-Specific Introduction of Chemically Reactive Handles in Proteins by UAA Mutagenesis

3 Luciferase as a Tool to Optimize the UAA Mutagenesis Method

4 Luciferase as a Tool to Evaluate Bioorthogonal Chemical Ligation Reactions to Modify UAAs

5 Synthesis of Probes with Reactive Groups for Bioorthogonal Reactions

6 Site-Specific Labeling of Membrane Proteins using Chemically Reactive Handles Introduced by Site-Directed UAA Mutagenesis

7 Extension of the Labeling Strategy to GPCRs of Unknown Structure

8 Engineering Constraints of Site-Specific Attachment of Fluorophores

9 Conclusions


Chapter Fourteen. Mapping a Ligand Binding Site Using Genetically Encoded Photoactivatable Crosslinkers

1 Introduction

2 Targeted Photocrosslinking in Live Cells Using Genetically Encoded Photocrosslinkers

3 Analysis of GPCR–Ligand Complexes

4 Conclusion


Chapter Fifteen. Alternative mRNA Splicing of G Protein-Coupled Receptors

1 Introduction

2 Establishment of Primary hMSMCs

3 Detection of Alternatively Spliced mRNA

4 Quantification of Alternatively Spliced mRNAs Using Real-Time qPCR

5 Summary


Chapter Sixteen. Functional Residues Essential for the Activation of the CB1 Cannabinoid Receptor

1 Introduction

2 The HU210-CB1 Receptor Model Representing an Early Stage of the Activated State

3 The HU210-CB1 Receptor in the Active State

4 Potential Functional Residues of the CB1 Receptor

5 Emerging Picture of the Molecular Mechanism of CB1 Receptor Activation


Author Index

Subject Index


This new volume of Methods in Enzymology continues the legacy of this premier serial by containing quality chapters authored by leaders in the field. This volume covers G protein coupled receptors and includes chapters on such topics as post-translation modification of GPCR in relationship to biased agonism, structure-based virtual screening, and GPCR oligomerization in the brain.

Key Features

  • Continues the legacy of this premier serial with quality chapters authored by leaders in the field
  • Covers G protein coupled receptors
  • Contains chapters on such topics as post-translation modification of GPCR in relationship to biased agonism, structure-based virtual screening, and GPCR oligomerization in the brain


Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists


No. of pages:
© Academic Press 2013
8th February 2013
Academic Press
Hardcover ISBN:
eBook ISBN:

Ratings and Reviews

About the Serial Volume Editor

P. Michael Conn

P. Michael Conn is the Senior Vice President for Research and Associate Provost, Texas Tech Health Sciences Center. He is The Robert C. Kimbrough, Professor of Internal Medicine and Cell Biology/Biochemistry. He was previously Director of Research Advocacy and Professor of Physiology and Pharmacology, Cell Biology and Development and Obstetrics and Gynecology at Oregon Health and Science University and Senior Scientist of the Oregon National Primate Research Center (ONPRC). He served for twelve years as Special Assistant to the President and Associate Director of the ONPRC. After receiving a B.S. degree and teaching certification from the University of Michigan (1971), a M.S. from North Carolina State University (1973), and a Ph.D. degree from Baylor College of Medicine (1976), Conn did a fellowship at the NIH, then joined the faculty in the Department of Pharmacology, Duke University Medical Center where he was promoted to Associate Professor in 1982. In 1984, he became Professor and Head of Pharmacology at the University of Iowa College of Medicine, a position he held for eleven years. Conn is known for his research in the area of the cellular and molecular basis of action of gonadotropin releasing hormone action in the pituitary and therapeutic approaches that restore misfolded proteins to function. His work has led to drugs that have benefitted humans and animals. Most recently, he has identified a new class of drugs, pharmacoperones, which act by regulating the intracellular trafficking of receptors, enzymes and ion channels. He has authored or co-authored over 350 publications in this area and written or edited over 200 books, including texts in neurosciences, molecular biology and endocrinology. Conn has served as the editor of many professional journals and book series (Endocrinology, Journal of Clinical Endocrinology and Metabolism, Endocrine, Methods, Progress in Molecular Biology and Translational Science and Contemporary Endocrinology). Conn served on the National Board of Medical Examiners, including two years as chairman of the reproduction and endocrinology committee. The work of his laboratory has been recognized with a MERIT award from the NIH, the J.J. Abel Award of the American Society for Pharmacology and Experimental Therapeutics, the Weitzman, Oppenheimer and Ingbar Awards of the Endocrine Society, the National Science Medal of Mexico (the Miguel Aleman Prize) and the Stevenson Award of Canada. He is the recipient of the Oregon State Award for Discovery, the Media Award of the American College of Neuropsychopharmacology and was named a distinguished Alumnus of Baylor College of Medicine in 2012. Conn is a previous member of Council for the American Society for Cell Biology and the Endocrine Society and is a prior President of the Endocrine Society, during which time he founded the Hormone Foundation and worked with political leadership to heighten the public’s awareness of diabetes. Conn’s students and fellows have gone on to become leaders in industry and academia. He is an elected member of the Mexican Institute of Medicine and a fellow of the American Association for the Advancement of Science. He is the co-author of The Animal Research War (2008) and many articles for the public and academic community on the value of animal research and the dangers posed by animal extremism. His op/eds have appeared in The Washington Post, The LA Times, The Wall Street Journal, the Des Moines Register, and elsewhere. Conn consults with organizations that are influenced by animal extremism and with universities and companies facing challenges from these groups.

Affiliations and Expertise

Texas Tech University Health Sciences Center, Lubbock, USA