Introduction

More than three decades of intensive research on GPCRs have brought about insights into some aspects of structure and function or membrane receptors, whereas many fundamental questions remain unresolved. Most of the unre-

*Present address: Pharmacology and Clinical Pharmacy, Philipps-Universitaet Marburg, Marburg, Germany

GPCR Molecular Pharmacology and Drug Targeting: Shifting Paradigms and New Directions,

Edited by Annette Gilchrist

Copyright © 2010 John Wiley & Sons, Inc.

solved questions are related to the dynamics of GPCR function, assembly, and signaling. Until 15 years ago, receptors could only be directly detected by means of radioligand binding assays and antibody binding. The function of receptors was determined either by measuring cellular responses resulting from the respective signaling pathway, or by means of radioactively labeled GTPyS binding to heterotrimeric G proteins.

The technical advancement which allowed labeling of receptors and proteins that interact with them using fluorescent tags revolutionized GPCR research as it enabled to image subcellular localization and to track internalization processes. One can now study the interaction of GPCRs with G proteins as well as with arrestins and can do so in a temporally resolved fashion, either indirectly by means of G protein activation or by monitoring conformational changes underlying receptor activation. A key to these developments was the discovery of fluorescent proteins [ 1, 2] , which were critically important for monitoring localization of receptors in living cells in real time. The ability to fuse fluorescent proteins to GPCRs or their interacting proteins accelerated the understanding of receptor trafficking and subcellular localization (see Section 10.3., Labeling GPCRs with Fluorescent Tags). The recent introduction of smaller fluorophores suitable for site-directed labeling of proteins in intact cells (see Section 10.3.2., Labeling of GPCRs with Fluorescent Dyes) accelerated functional studies on GPCRs [3-5]. A major breakthrough for investigation of receptor assemblies, as well as dynamics of receptor function in living cells, came with the introduction of variants of GFP suitable for FRET [6] and, similarly, the establishment of luciferase/GFP variants useful for BRET [7] , which allowed addition of optical "rulers" with which to measure receptors and their interacting binding partners (see Section 10.6., Resonance Energy Transfer, a Tool to Get New Insight into GPCR Function). FRET and BRET assays are extremely sensitive and can be used to measure changes in distance close to the Förster radius (typically between 4 nm and 6 nm). This distance is optimal for studying the interaction between small- to medium - size proteins and also allows detection of conformational changes within a protein that lead to significant intramolecular movements. In addition to the different labeling approaches in this chapter, we discuss BRET and FRET as methods to study interactions of receptors and their function in living cells. A special emphasis will be the potential pitfalls and problems in the practical use of these methods.

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