G Protein-Coupled Receptor Screening Assays: Methods and Protocols

ISBN-13: 9781071612231 / Angielski / Miękka / 2022

Martins, Sofia Aires M.

Opis

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.

Spis treści

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 Salmerón, 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 Köck, Volker Dötsch, 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, Tõnis 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

            Céline 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, Tõnis 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-Gardón and Raúl Estévez

 

16. NanoLuc-Based Methods to Measure β-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 João P. Conde

 

20. Homology Modeling Using GPCRM Web Service

            Przemysław Miszta, Szymon Niewieczerzał, Paweł Pasznik, and Sławomir Filipek