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G Protein-Coupled Receptor Screening Assays
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This volume explores the considerable efforts that have been directed towards the development of G Protein-Coupled Receptors (GPCR) screening assays in order to disclose GPCR acting compounds, elucidate signaling mechanisms or evaluate compound's efficacy. New discoveries in the field, along with the widely recognized need for better and safer pharmaceutical drugs constitute the main motivation for this book. Readers, both beginners and experienced researchers, will receive an updated overview of not only the established, but also the innovative technologies that promise to advance GPCR drug research. This book is organized into two major parts: the introductory part discusses the necessary foundations for the understanding of GPCR action and the rationale behind the design of the available screening assays; and part two provides detailed protocols for different screening approaches. Written in the highly successful Methods in Molecular Biology series format, the chapters include the kind of detailed description and implementation advice that is crucial for getting optimal results in the laboratory. Practical and innovative, G Protein-Coupled Receptor Screening Assays: Methods and Protocols reaches out to everyone involved in the discovery of GPCR-active drugs, and provides a transversal overview of the different levels of GPCR signaling addressable in the different screening strategies and presents practical examples of how current assay technologies are contributing to new paradigms in GPCR drug research.
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Table of Contents

Part I: Introduction 1. G-Protein Coupled Receptors: An Overview of Signaling Mechanisms and Screening Assays Duarte Miguel F. Prazeres and Sofia Aires M. Martins Part II: GPCR Screening Assays 2. Time-Resolved FRET Strategy to Screen GPCR Ligands Library Nadia Oueslati, Candide Hounsou, Abderazak Belhocine, Thieric Rodriguez, Elodie Dupuis, Jurriaan M. Zwier, Eric Trinquet, Jean-Philippe Pin, and Thierry Durroux 3. Homogeneous Fluorescence Anisotropy-Based Assay for Characterization of Ligand Binding Dynamics to GPCRs in Budded Baculoviruses: The Case of Cy3B-NDP-a-MSH Binding to MC4 Receptors Santa Veiksina, Sergei Kopanchuk, Olga Mazina, Reet Link, Anne Lille, and Ago Rinken 4. Construction of Recombinant HEK293 Cell Lines for the Expression of the Neurotensin Receptor NTSR1 Su Xiao, Joseph Shiloach, and Reinhard Grisshammer 5. cAMP Assay for GPCR Ligand Characterization: Application of BacMam Expression System Olga Mazina, Anni Allikalt, Annika Heinloo, Reet Reinart-Okugbeni, Sergei Kopanchuk, and Ago Rinken 6. Ca2+ Mobilization Assays in GPCR Drug Discovery Grzegorz Woszczek and Elisabeth Fuerst 7. Using Constitutive Activity to Define Appropriate High Throughput Screening Assays for Orphan G Protein-Coupled Receptors Tony Ngo, James L. Coleman, and Nicola J. Smith 8. Monitoring G Protein-Coupled Receptor Activation Using the Protein Fragment Complementation Technique Split TEV Michael C. Wehr, Sabrina Galinski, and Moritz J. Rossner 9. Quantifying GPCR Internalization: A Focus on the Kisspeptin Receptor Macarena Pampillo and Andy V. Babwah 10. GPCR Oligomerization Analysis by Means of BRET and dFRAP Francisco Ciruela and Victor Fernandez-Duenas 11. Use of ImageJ to Recover Information from Individual Cells in a G-Protein Coupled Receptor Assay J. R. C. Trabuco, Sofia Aires M. Martins, and Duarte Miguel F. Prazeres 12. Methods to Immobilize GPCR on the Surface of SPR Sensors Laura Martinez-Munoz, Ruben Barroso, Anabel Guedan Paredes, Mario Mellado, and Jose Miguel Rodriguez-Frade 13. Olfactory Receptor Screening Assay Using Nanovesicle-Immobilized Carbon Nanotube Transistor Jong Hyunn Lim, Juhum Park, Seunghun Hong, and Tai Hyun Park 14. Label-Free Biosensor Assays in GPCR Screening Manuel Grundmann and Evi Kostenis 15. Multi-Dimensional GPCR Profiling and Screening Using Impedance-Based Label-Free and Real-Time Assay Ning Ke, Khanh Nguyen, Jeffrey Irelan, and Yama A. Abassi 16. Label-Free Functional Selectivity Assays Ann M. Ferrie, Vasiliy Goral, Chaoming Wang, and Ye Fang 17. Measurement of Surface-Mediated Ca++ Transients on the Single-Cell Level in a Microfluidic Lab-on-a-chip Environment Michael Kirschbaum, Magnus S. Jaeger, and Claus Duschl 18. Cell-Based Assays and Animal Models for GPCR Drug Screening Hideo Takakura, Mitsuru Hattori, Miho Tanaka, and Takeaki Ozawa 19. Computer-Aided Design of GPCR Ligands Hugo Gutierrez-de-Teran, Henrik Keranen, Jhonny Azuaje, David Rodriguez, Johan Aqvist, Eddy Sotelo

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