Time-Resolved Förster Resonance Energy Transfer-Based Technologies to Investigate G Protein-Coupled Receptor Machinery: High-Throughput Screening Assays and Future Development - Progress in Molecular Biology and Translational Science - page 274

CHAPTER SEVEN

Time-Resolved Förster Resonance
Energy Transfer-Based
Technologies to Investigate
G Protein-Coupled Receptor
Machinery: High-Throughput
Screening Assays and Future
Development

, , ,}

, Jurriaan M. Zwier } , Eric Trinquet } ,

† ‡

Pauline Scholler *

, , , , , ,

† ‡ , Jean-Philippe Pin * † ‡ , Laurent Prézeau * † ‡ ,

Philippe Rondard *

, ,

‡
* CNRS, UMR-5203, Institut de G ´ nomique Fonctionelle, Montpellier, F-34094, France
† INSERM, U661, Montpellier, F-34094, France
‡

†

Julie Kniazeff *

Universit ´ s de Montpellier 1&2, UMR-5203, Montpellier, F-34094, France
} Cisbio Bioassays, Parc Marcel Boiteux, BP 84175, Codolet, France

Contents
1. Introduction 276

2. Overview of the TR-FRET Principle and Its Advantages 278

2.1 Principle of FRET 279

2.2 Principle of TR-FRET 279

2.3 Advantages of TR-FRET over classic FRET 280

2.4 TR-FRET in HTS campaigns 282

3. Screening with Generic Methods 283

3.1 Sandwich or competitive assay format 283

3.2 G protein activation and secondary messengers 285

3.3 Later signaling events 290

3.4 The future of HTS generic assays 292

4. Screening with Target-Specific Methods 293

4.1 Drugs targeting a protein: Ligand-binding assay 293

4.2 Drugs targeting a protein complex 297

5. Perspectives 305

5.1 Detecting GPCR conformational changes upon binding 305

5.2 Detecting GPCR internalization 306