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G Protein Coupled Receptors
 
 

G Protein Coupled Receptors, 1st Edition

Trafficking and Oligomerization

 
G Protein Coupled Receptors, 1st Edition,P. Michael Conn,ISBN9780123918628
 
 
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Methods in Enzymology

P Conn   

Academic Press

9780123918628

9780123918734

480

229 X 152

This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. It covers G protein coupled receptors and includes chapters on such topics as G protein-coupled receptor trafficking motifs, structure-based virtual screening, and automation-friendly high-throughput assays for identifying pharmacoperone drugs.

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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 G protein-coupled receptor trafficking motifs, structure-based virtual screening, and automation-friendly high-throughput assays for identifying pharmacoperone drugs

Description

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 G protein-coupled receptor trafficking motifs, structure-based virtual screening, and automation-friendly high throughput assays for identification of pharmacoperone drugs.

Readership

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

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

View additional works by P. Michael Conn

G Protein Coupled Receptors, 1st Edition

Series Page

Contributors

Preface

Methods in Enzymology

Chapter One. Therapeutic Rescue of Misfolded/Mistrafficked Mutants

1. Introduction

2. Choosing Pharmacoperone Model Systems

3. Selection of Endpoint Measures

4. Assay Automation

5. Data Analysis

6. Hit Follow-Up Experiments

7. Conclusions

8. Acknowledgments

References

Chapter Two. Trafficking of the Follitropin Receptor

1. Introduction

2. Outward Trafficking Defective FSHR Mutants. Studying Plasma Membrane Expression of the FSHR

3. Studying Oligomerization of Intracellular and Cell Surface-Expressed FSHRs

4. Phosphorylation, Internalization, and Recycling of the FSHR (Downward Trafficking)

5. Acknowledgments

References

Chapter Three. Single-Molecule Imaging Technique to Study the Dynamic Regulation of GPCR Function at the Plasma Membrane

1. Introduction

2. Labeling of GPCRs and Sample Preparation for SPT

3. Single-Molecule Imaging

4. Data Analysis

5. Concluding Remarks

6. Acknowledgments

References

Chapter Four. GPCR Oligomerization and Receptor Trafficking

1. Introduction

2. GPCR Expression Using the Flp-In™ T-Rex™ System

3. Detecting GPCR Internalization by Fluorescent Microscopy: The Orexin OX1 Receptor

4. SNAP–CLIP Tagging

5. Detecting GPCR Oligomerization by other Resonance Energy Transfer Techniques

6. Biotinylation Studies

7. ER Trapping, Pharmacological Chaperones, and the Use of Engineered Synthetic Ligands

8. Acknowledgments

References

Chapter Five. β-Arrestins and G Protein-Coupled Receptor Trafficking

1. Introduction

2. Arrestin Expression

3. Assays to Measure GPCR Trafficking

4. Evaluating the Role of β-Arrestins in GPCR Trafficking

5. Summary

6. Acknowledgments

References

Chapter Six. Tracking Cell Surface Mobility of GPCRs Using α-Bungarotoxin-Linked Fluorophores

1. Introduction

2. Methodology

3. Validating the BBS Tag

4. Experimental Applications for the BBS Tag

5. Image Analysis

6. Other Applications

7. Overview

References

Chapter Seven. Regulatory Mechanism of G Protein-Coupled Receptor Trafficking to the Plasma Membrane

1. Introduction

2. Purification and Identification of β2-AR mRNA-Binding Proteins

3. Functional Characterization of β2-AR mRNA-Binding Proteins in Receptor Expression and Function

4. Role of RNA-Binding Protein HuR in Receptor Trafficking to the Plasma Membrane

5. β2-AR mRNA Localization in Cells

6. Concluding Remarks and Future Perspectives

7. Acknowledgments

References

Chapter Eight. Dissecting Trafficking and Signaling of Atypical Chemokine Receptors

1. Introduction

2. ACR Trafficking

3. Atypical Chemokine Receptor Signaling

4. Summary

References

Chapter Nine. Systematic and Quantitative Analysis of G Protein-Coupled Receptor Trafficking Motifs

1. Introduction

2. Motif Screening by Immunofluorescent Staining

3. Analysis of Receptor Functionality

4. Biochemical Analysis of ER/Golgi Trafficking

5. Quantitative Analysis of GPCR Trafficking

6. Summary

7. Acknowledgments

References

Chapter Ten. Identification of Endoplasmic Reticulum Export Motifs for G Protein-Coupled Receptors

1. Introduction

2. Experimental Approaches to Identify ER Export Motifs for GPCRs

3. Conclusions

4. Acknowledgment

References

Chapter Eleven. Amino Acid Residues of G-Protein-Coupled Receptors Critical for Endoplasmic Reticulum Export and Trafficking

1. Introduction

2. Generation of Mutant GPCRs

3. Examination of Mutant GPCR Trafficking

4. ER Export of Mutant GPCRs by Specific Ligands

5. Functional Analysis of Surface-Trafficked Mutant GPCRs in Living Cells

6. Conclusion

Acknowledgments

References

Chapter Twelve. G-Protein-Coupled Heteromers

1. Introduction

2. Generation of Heteromer-Selective mAbs

3. ELISA for Detection of Receptor Heteromers

4. Immunofluorescence for Visualization of Receptor Heteromers

5. Immunoprecipitation and Western Blotting

6. Summary and Perspectives

7. Acknowledgment

References

Chapter Thirteen. Hetero-oligomerization and Specificity Changes of G Protein-Coupled Purinergic Receptors

1. Introduction

2. Measurement of GPCR Dimerization

3. Receptor Pharmacology

4. Conclusion

References

Chapter Fourteen. Bimolecular Fluorescence Complementation Analysis of G Protein-Coupled Receptor Dimerization in Living Cells

Abbreviations

1. Introduction

2. Generation of GPCR–BiFC Fusion Proteins

3. Detection of GPCR Interactions using BiFC and Fluorescence Microscopy

4. Microscopic Detection of GPCR Interactions by mBiFC

5. Fluorometric Detection of GPCR Dimerization using BiFC and mBiFC

6. Summary

7. Acknowledgments

References

Chapter Fifteen. G Protein–Coupled Receptor Heterodimerization in the Brain

1. Introduction

2. In Situ PLA for Demonstrating Receptor Heteromers and Their Receptor–Receptor Interactions in Brain Tissue

3. Brain Tissue Preparation

4. Proximity Probes: Conjugation of Oligonucleotides to Antibodies

5. PLA Reactions, Reagents, and Solutions

6. Quantitative PLA Image Analysis

7. Advantages and Disadvantages of the PLA Method

8. Application

9. Acknowledgments

References

Chapter Sixteen. Experimental Strategies for Studying G Protein-Coupled Receptor Homo- and Heteromerization with Radioligand Binding and Signal Transduction Methods

1. Introduction

2. Radioligand Binding in Receptor Oligomerization

3. Signal Transduction in Receptor Oligomerization

4. Domain Swapping in Receptor Oligomerization

5. Concluding Remarks

References

Chapter Seventeen. Analysis of GPCR Dimerization Using Acceptor Photobleaching Resonance Energy Transfer Techniques

1. Introduction

2. Resonance Energy Transfer

3. Fluorescent Resonance Energy Transfer

4. BRET

5. Conclusions

6. Acknowledgments

References

Chapter Eighteen. Techniques for the Discovery of GPCR-Associated Protein Complexes

1. Introduction

2. TAP of GPCR and Its Associated Protein Complexes

3. Alternative Methodology to Perform TAP of GPCR-Associated Protein Complexes

4. Purification of GPCR-Associated Protein Complexes by Peptide Affinity Chromatography

5. Conclusion

6. Acknowledgments

References

Chapter Nineteen. Expression, Purification, and Analysis of G-Protein-Coupled Receptor Kinases

1. Introduction

2. Expression and Purification of GRKs

3. GRK Functional Assays

4. Acknowledgments

References

Chapter Twenty. Modern Methods to Investigate the Oligomerization of Glycoprotein Hormone Receptors (TSHR, LHR, FSHR)

1. Introduction

2. Resonance Energy Transfer Techniques

3. Experimental Procedures

4. Acknowledgments

References

Author Index

Subject Index

 
 
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