G Protein Coupled Receptors

Contributors

Preface

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

References

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

References

Chapter Three. Molecular Modeling of Adenosine Receptors

1 Introduction

2 Adenosine A2a Receptor

3 Receptor Modeling

4 Summary

References

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

References

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

References

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

References

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

References

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

References

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

References

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

References

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

References

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

References

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

References

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

References

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

References

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

References

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

References

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

Abbreviations

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

References

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

References

Author Index

Subject Index