G Protein Coupled Receptors, Volume 521

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

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.

• 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

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