G Protein Coupled Receptors

G Protein Coupled Receptors

Modeling, Activation, Interactions and Virtual Screening

1st Edition - January 28, 2013

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  • Editor: P. Michael Conn
  • eBook ISBN: 9780124079441
  • Hardcover ISBN: 9780124078659

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This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers G protein coupled receptors, and includes chapters on such topics as GPCR modelling, interactions with other molecules, virtual screening and GPCR activation.

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 GPCR modelling, interactions with other molecules, virtual screening and GPCR activation


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

Table of Contents

  • Contributors


    Methods in Enzymology

    Chapter One. The Protein Local Optimization Program and G-Protein-Coupled Receptors: Loop Restoration and Applications to Homology Modeling

    1 Introduction

    2 PLOP Performance for Loop Prediction

    3 PLOP and GPCRs

    4 Loop Prediction with the Single-Residue Library

    5 Loop Prediction with the Dipeptide Library

    6 Hierarchical Loop Prediction with Surrounding Side Chain Optimization

    7 Additional Sampling Methods

    8 VSGB 2.0: The Newest Energy Model Incorporated into PLOP

    9 Conclusions


    Chapter Two. Modeling Active GPCR Conformations

    1 Introduction

    2 General Approaches

    3 Agonist-Induced Activation

    4 Simulation Methods

    5 Microswitches

    6 Biophysical Restraints

    7 Virtual Screening

    8 Class B GPCRs


    Chapter Three. Molecular Modeling of Adenosine Receptors

    1 Introduction

    2 Adenosine A2a Receptor

    3 Receptor Modeling

    4 Summary


    Chapter Four. Predicting G-Protein-Coupled Receptors Families Using Different Physiochemical Properties and Pseudo Amino Acid Composition

    1 Introduction

    2 GPCRs Prediction

    3 Performance Evaluation of GPCR Predictor

    4 Webserver for GPCR Predictor

    5 Concluding Remarks


    Chapter Five. Computational Design and Experimental Characterization of GPCR Segment Models

    1 Introduction

    2 Design of GPCR Segment Models

    3 Experimental Characterization of GPCR Segment Models

    4 Integration of GPCR Segment Models into GPCR Homology Models

    5 Summary


    Chapter Six. G-Protein-Coupled Receptor Interaction with Small GTPases

    1 Introduction

    2 Assays for GPCR Interaction with Small GTPases

    3 Function of GPCR–Small GTPase Interaction


    Chapter Seven. Quantification of Receptor Tyrosine Kinase Activation and Transactivation by G-Protein-Coupled Receptors Using Spatial Intensity Distribution Analysis (SpIDA)

    1 Introduction

    2 Theory of Spatial Intensity Distribution Analysis

    3 Approach Validation

    4 Procedure for SpIDA

    5 Conclusions


    Chapter Eight. NMR Methods for Detection of Small Molecule Binding to RGS4

    1 Introduction

    2 Limitations and Challenges of NMR Experiments on RGS Proteins

    3 Construct Selection for NMR Studies with RGS4

    4 Expression and Purification of 15N-Labeled Δ51 RGS4

    5 Setting Up a Protein-Based NMR Fragment Screen Targeting Δ51 RGS4

    6 Alternative Ligand-Based Approach Using Saturation Transfer Difference for Small Molecule Studies with Unlabeled RGS

    7 Conclusion


    Chapter Nine. Group II Activators of G-protein Signaling: Monitoring the Interaction of Ga with the G-protein Regulatory Motif in the Intact Cell

    1 Introduction

    2 Generation of Probes for Measurement of GPR–Gαi Interaction in Cells by BRET

    3 Expression of Donor and Acceptor in Cells

    4 Analysis of GPR–Gαi1 Interaction by BRET

    5 Regulation of the GPR–Gαi Complex by a G-protein-Coupled Receptor

    6 Analysis of GPR–Gαi1 Interaction in Subcellular Fractions by BRET

    7 Summary


    Chapter Ten. Homogeneous Time-Resolved Fluorescence Assay to Probe Folded G Protein-Coupled Receptors

    1 Introduction

    2 Solid-Phase Labeling of IgG with Europium Cryptate

    3 HTRF Assay Standards and Signal Analysis

    4 Applications of GPCR HTRF Assay

    5 Conclusion


    Chapter Eleven. cAMP Biosensors Applied in Molecular Pharmacological Studies of G Protein-Coupled Receptors

    1 Introduction

    2 Generation of an Epac1-Based cAMP Biosensor Cell Line with an Optimized Fluorescent Protein FRET Pair

    3 cAMP Biosensor Characterization

    4 Assay for Studying GPCR Signaling as Exemplified by Gs-Coupled β2-Adrenergic Receptor Activation

    5 Modifications to the General cAMP Kinetics Assay


    Chapter Twelve. Rhodopsin–Lipid Interactions Studied by NMR

    1 Introduction

    2 Reconstitution of GPCR into Single, Tubular Bilayers Using Nanoporous Substrates

    3 Nuclear Magnetic Resonance

    4 Conclusions


    Chapter Thirteen. Biasing the Parathyroid Hormone Receptor: Relating In Vitro Ligand Efficacy to In Vivo Biological Activity

    1 Introduction

    2 Determining the Relative Activity of PTH1R Ligands

    3 Discussion


    Chapter Fourteen. Application of Monte Carlo-Based Receptor Ensemble Docking to Virtual Screening for GPCR Ligands

    1 Introduction

    2 Description of the Methodology

    3 A Practical Example: Application of Monte Carlo-Based RED to a Controlled Retrospective Virtual Screening for β2 Adrenergic Receptor Ligands

    4 Final Considerations


    Chapter Fifteen. From Heptahelical Bundle to Hits from the Haystack

    1 From Heptahelical Bundles to Hits from the Haystack: Structural Molds for Virtual Screening

    2 Shaping Structures, Carving Cavities: A Customized Structure-Based GPCR Modeling Workflow

    3 In Silico Screening Success Stories: From GPCR Structure to Novel GPCR Ligands

    4 Navigating Novel GPCR–Ligand Interaction Space


    Chapter Sixteen. Kinetics and Dynamics in the G Protein-Coupled Receptor Signaling Cascade

    1 Introduction

    2 FRET Assays to Measure Reaction Kinetics in GPCR Signaling Cascades

    3 Dynamic of Receptor–Protein Interactions

    4 ICCS to Determine GPCR Dynamics


    Chapter Seventeen. Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy of G Protein-Coupled Receptors

    1 Introduction

    2 Large-Scale GPCR Expression in HEK293 Cells

    3 Reconstitution of GPCRs

    4 MAS NMR Spectroscopy of GPCRs


    Chapter Eighteen. Posttranslation Modification of G Protein-Coupled Receptor in Relationship to Biased Agonism


    1 Introduction

    2 Posttranslation Modification of GPCR with Biased Signaling

    3 Membrane Distribution of GPCR with Biased Signaling

    4 Possible Interaction Between These Three Methods

    5 Future Direction of Biased Signaling


    Chapter Nineteen. Protein S-Nitrosylation Measurement

    1 Introduction

    2 Biotin Switch Technique

    3 Fluorescence Switch Technique

    4 His-Tag Switch Technique

    5 In Situ BST

    6 SNO-RAC

    7 Mass Spectrometry Analysis

    8 NO Detection Methods

    9 S—NO Antibody

    10 Summary


    Author Index

    Subject Index

Product details

  • No. of pages: 528
  • Language: English
  • Copyright: © Academic Press 2013
  • Published: January 28, 2013
  • Imprint: Academic Press
  • eBook ISBN: 9780124079441
  • Hardcover ISBN: 9780124078659

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

Senior Vice President for Research and Associate Provost, Texas Tech Health Sciences Center, TX, USA

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