Main description:

This detailed volume assembles comprehensive protocols to assist with the study of structural, molecular, cell biological, and in vivo facets of GPCRs, and to enable the development of experimental tools for screening novel GPCR drugs. Sections explore the tweaking of ligands, bioluminescence and FRET approaches, specific GPCR signaling properties, as well as visualization of subcellular compartmentalization. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, G Protein-Coupled Receptor Signaling: Methods and Protocols serves as an ideal reference for life scientists working in a variety of research fields including molecular pharmacology, cell and developmental biology, brain behavior and physiology, drug development and screening.
Chapter 4 is available open access under a CC BY 4.0 license via link.springer.com.

Contents:

Part I: Probing Structural Properties of GPCRs

1. Assessment of Conformational State Transitions of Class B GPCRs Using Molecular Dynamics

Chenyi Liao, Victor May, and Jianing Li

2. Molecular Dynamic Simulations to Probe Water Permeation Pathways of GPCRs

Katsufumi Tomobe, Eiji Yamamoto, and Kenji Yasuoka

3. Expression and Purification of a Functional E. coli 13CH3-Methionine-Labeled Thermostable Neurotensin Receptor 1 Variant for Solution NMR Studies

Fabian Bumbak, Ross A.D. Bathgate, Daniel J. Scott, and Paul R. Gooley

4. A Combined Cell-Free Protein Synthesis and Fluorescence-Based Approach to Investigate GPCR Binding Properties

Anne Zemella, Theresa Richter, Lena Thoring, and Stefan Kubick

Part II: Tweaking Ligands for GPCR Studies

5. Furan Cross-Linking Technology for Investigating GPCR-Ligand Interactions

Marleen Van Troys, Willem Vannecke, Christophe Ampe, and Annemieke Madder

6. Optical Regulation of Class C GPCRs by Photoswitchable Orthogonal Remotely-Tethered Ligands

Amanda Acosta-Ruiz, Johannes Broichhagen, and Joshua Levitz

7. Chemoselective Acylation of Hydrazinopeptides to Access Fluorescent Probes for Time-Resolved FRET Assay on GPCRs

Sridevi M. Ramanoudjame, Lucie Esteoulle, Stephanie Riche, Jean-Francois Margathe, Thierry Durroux, Iuliia A. Karpenko, and Dominique Bonnet

Part III: Bioluminescence and Fluorescence Resonance Transfer Energy Approaches

8. Time-Resolved FRET-Based Assays to Characterize G Protein-Coupled Receptor Hetero-Oligomer Pharmacology

Joyce Heuninck, Candide Hounsou, Elodie Dupuis, Eric Trinquet, Bernard Mouillac, Jean-Philippe Pin, Dominique Bonnet, and Thierry Durroux

9. Combining Conformational Profiling of GPCRs with CRISPR/Cas9 Gene Editing Approaches

Kyla Bourque, Dominic Devost, Asuka Inoue, and Terence E. Hebert

10. Measuring GPCR Stoichiometry Using Types -1, -2 and -3 Bioluminescence Resonance Energy Transfer-Based Assays

James H. Felce, John R. James, and Simon J. Davis

11. Combining SRET2 and BiFC to Study GPCR Heteromerization and Protein-Protein Interactions

Amina M. Bagher, Melanie E.M. Kelly, and Eileen M. Denovan-Wright

12. Quantification and Comparison of Signals Generated by Different FRET-Based cAMP Reporters

Andreas Koschinski and Manuela Zaccolo

Part IV: Exploring GPCR Signaling Properties

13. Measuring GPCR-Induced Activation of Protein Tyrosine Phosphatases (PTP) Using In-Gel and Colorimetric PTP Assays

Genevieve Hamel-Cote, Fanny Lapointe, and Jana Stankova

14. Measurement of -Arrestin Recruitment at GPCRs Using the Tango Assay

Genevieve Laroche and Patrick M. Giguere

15. Probing the Interactome of Corticotropin-Releasing Factor Receptor Heteromers Using Mass Spectrometry

Burcu Hasdemir, Juan A. Oses-Prieto, Alma Burlingame, and Aditi Bhargava

Part V: Visualizing Subcellular Compartmentalization of GPCRs

16. Monitoring the Aggregation of GPCRs by Fluorescence Microscopy

Samuel Genier, Jade Degrandmaison, Christine L. Lavoie, Louis Gendron, and Jean-Luc Parent

17. Combining RNAi and Immunofluorescence Approaches to Investigate Post-Endocytic Sorting of GPCRs into Multivesicular Bodies

Xuezhi Li, Stephanie Rosciglione, Andreanne Laniel, and Christine Lavoie

18. Super-Resolution Imaging of G Protein-Coupled Receptors Using Ground State Depletion Microscopy

Fabiana A. Cateano-Crowley, Bryan Heit, and Stephen S.G. Ferguson

19. Analysis of Spatial Assembly of GPCRs Using Photoactivatable Dyes and Localization Microscopy

Kim C. Jonas and Aylin C. Hanyaloglu

Part VI: Investigating In Vivo Functions of GPCRs

20. Optical Modulation of Metabotropic Glutamate Receptor Type 5 In Vivo Using a Photoactive Drug

Marc Lopez-Cano, Joan Font, Amadeu Llebaria, Victor Fernandez-Duenas, and Francisco Ciruela

21. Imaging of Tissue-Specific and Temporal Activation of GPCR Signaling Using DREADD Knock-In Mice

Dmitry Akhmedov, Nicholas S. Kirkby, Jane A. Mitchell, and Rebecca Berdeaux

22. Laser Doppler Flowmetry to Study the Regulation of Cerebral Blood Flow by G Protein-Coupled Receptors in Rodents

Xavier Toussay, Mario Tiberi, and Baptiste Lacoste

23. Preparation of Pulmonary Artery Myocytes and Rings to Study Vasoactive GPCRs

Martha Hinton, Anurag Singh Sikarwar, and Shyamala Dakshinamurti