G Protein-Coupled Receptor Screening Assays

1. Front Matter

   Pages i-xvi

   PDF

2. Isolation of Lipid Rafts by the Detergent-Based 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

   Pages 1-20

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

   • Krishna Sriram, Cristina Salmerón, Anna Di Nardo, Paul A. Insel

   Pages 21-42

4. Construction of Recombinant Cell Lines for GPCR Expression

   • Philip J. Reeves

   Pages 43-60

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

   • Alexei A. Yeliseev

   Pages 61-76

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

   • Emily A. Yasi, Pamela Peralta-Yahya

   Pages 77-84

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

   • Jasmina Vidic, Yanxia Hou

   Pages 85-95

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

   • Zoe Köck, Volker Dötsch, Frank Bernhard

   Pages 97-117

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

   • Santa Veiksina, Maris-Johanna Tahk, Tõnis Laasfeld, Reet Link, Sergei Kopanchuk, Ago Rinken

   Pages 119-136

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

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

    Pages 137-147

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

    • Céline Laschet, Julien Hanson

    Pages 149-157

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

    • Luca Bordes, Sergei Chavez-Abiega, Joachim Goedhart

    Pages 159-178

13. 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 et al.

    Pages 179-192

14. FLIPR Calcium Mobilization Assays in GPCR Drug Discovery

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

    Pages 193-205

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

    • Xenia Meshik, Narasimhan Gautam

    Pages 207-221

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

    • Marta Alonso-Gardón, Raúl Estévez

    Pages 223-232

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

    • Xiaoyuan Ma, Rob Leurs, Henry F. Vischer

    Pages 233-248

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

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

    Pages 249-274

19. Gradient Tracking by Yeast GPCRs in a Microfluidics Chamber

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

    Pages 275-287

20. 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, João P. Conde

    Pages 289-304

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.

• GPCRs
• Drug target candidates
• Drug design
• Receptor purification
• Reporter systems
• Recombinant engineering

• INESC Microsistemas e Nanotecnologias, Lisbon, Portugal

  Sofia Aires M. Martins

• IBB—Institute for Bioengineering and Biosciences, Lisbon, Portugal

  Duarte Miguel F. Prazeres

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