G Protein Coupled Receptors

Series Page

Contributors

Preface

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

References

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

References

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

Abbreviations

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

References

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

1 Introduction

2 Methods

References

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

References

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

References

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

References

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

References

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

References

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

Abbreviations

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

References

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

References

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

Abbreviations

1 Introduction

2 Synthesis of mGluR-CT

3 Structural Analysis

References

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

References

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

References

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

References

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

References

Author Index

Subject Index