Main description:

This fully updated edition targets not only those assays directly involved in the discovery of GPCR-active compounds but also those involved in cell-based experiments designed to study physiological responses. Whether coming from academia or industry, or being an experienced researcher or a newcomer to the field, the reader will find accessible methods and protocols that cover the latest developments on receptor purification, molecular biology, recombinant engineering, and analytical techniques that enable the real time monitoring of the complex GPCR signaling cascade and identification of potential drug targets. 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 up-to-date, G Protein-Coupled Receptor Screening Assays: Methods and Protocols, Second Edition aims to provide the tools necessary to contribute to the advancement of GPCR research and discovery and ultimately lead to the availability of innovative and more efficient drugs.

Contents:

1. Isolation of Lipid Rafts by the Detergent and Non-Detergent Based Methods for Localization of GPCRs with Immunoblotting and Laser Scanning Confocal Microscopy

Peter Abdelmaseeh, Andrew C. Tiu, Selim Rozyyev, Laureano D. Asico, Pedro A. Jose, Van Anthony M. Villar

2. Detection of GPCR mRNA Expression in Primary Cells via qPCR, Microarrays, and RNA-Sequencing

Krishna Sriram, Cristina Salmeron, Anna Di Nardo, and Paul A. Insel

3. Construction of Recombinant Cell Lines for GPCR Expression

Philip J. Reeves

4. Recombinant Expression and Purification of Cannabinoid Receptor CB2, a G Protein-Coupled Receptor

Alexei A. Yeliseev

5. Screening for Serotonin Receptor 4 Agonists Using GPCR-Based Sensor in Yeast

Emily A. Yasi and Pamela Peralta-Yahya

6. Immobilization of Olfactory Receptors Carried by Nanosomes onto a Gold Sensor Surface

Jasmina Vidic and Yanxia Hou

7. Screening Methods for Cell-Free Synthesized GPCR/Nanoparticle Samples

Zoe Koeck, Volker Doetsch, and Frank Bernhard

8. Fluorescence Anisotropy-Based Assay for Characterization of Ligand Binding Dynamics to GPCRs: The Case of Cy3B-Labelled Ligands Binding to MC4 Receptors in Budded Baculoviruses

Santa Veiksina, Maris-Johanna Tahk, Tonis Laasfeld, Reet Link, Sergei Kopanchuk, and Ago Rinken

9. Bioluminescence in G Protein-Coupled Receptors Drug Screening Using Nanoluciferase and Halo-Tag Technology

Hannes Schihada, Katarina Nemec, Martin J. Lohse, and Isabella Maiellaro

10. Nanoluciferase-Based Complementation Assay to Detect GPCR-G Protein Interaction

Celine Laschet and Julien Hanson

11. Imaging of Genetically-Encoded FRET-Based Biosensors to Detect GPCR Activity

Luca Bordes, Sergei Chavez-Abiega, and Joachim Goedhart

12. cAMP Biosensor Assay Using BacMam Expression System: Studying the Downstream Signaling of LH/hCG Receptor Activation

Darja Lavogina, Tonis Laasfeld, Maris-Johanna Tahk, Olga Kukk, Anni Allikalt, Sergei Kopanchuk, and Ago Rinken

13. FLIPR Calcium Mobilization Assays in GPCR Drug Discovery

Grzegorz Woszczek, Elisabeth Fuerst, and Thomas J.A. Maguire

14. Live Cell Imaging and Optogenetics-Based Assays for GPCR Activity

Xenia Meshik and N. Gautam

15. Split-Tobacco Etch Virus (Split-TEV) Method in G Protein-Coupled Receptor Interacting Proteins

Marta Alonso-Gardon and Raul Estevez

16. NanoLuc-Based Methods to Measure -Arrestin2 Recruitment to G Protein-Coupled Receptors

Xiaoyuan Ma, Rob Leurs, and Henry F. Vischer

17. Luciferase Complementation Approaches to Measure GPCR Signaling Kinetics and Bias

Nicola C. Dijon, Desislava N. Nesheva, and Nicholas D. Holliday

18. Gradient Tracking by Yeast GPCRs in a Microfluidics Chamber

Sara Kimiko Suzuki, Joshua B. Kelley, Timothy C. Elston, and Henrik G. Dohlman

19. Monitoring Intracellular Calcium in Response to GPCR Activation: Comparison Between Microtiter Plates and Microfluidic Assays

Sofia Aires M. Martins, Duarte Miguel F. Prazeres, Virginia Chu, and Joao P. Conde

20. Homology Modeling Using GPCRM Web Service

Przemyslaw Miszta, Szymon Niewieczerzal, Pawel Pasznik, and Slawomir Filipek