Photobleaching fluorescence resonance energy transfer reveals ligand-induced oligomer formation of human somatostatin receptor subtypes

The existence of dimers and higher oligomers of G-protein-coupled receptors (GPCRs) has been frequently reported using strategies based on coimmunoprecipitation or Western blot assays. These methods rely on highly artificial systems with over-expressed receptors, resulting in conflicting observations on the question of whether GPCR dimers are preformed or are formed in response to agonist treatment. Fluorescence resonance energy transfer (FRET) microscopy is a superior and less perturbing technique which can be performed on selected cell regions, e.g., plasma membrane of intact cells with a sensitivity high enough to allow study under physiological levels of receptor expression. Here we describe the application of photobleaching (pb) FRET microscopy for investigating ligand-dependent oligomerization of somatostatin receptors. Procedures for the introduction of suitable donor-acceptor fluorophores in a given GPCR are described. The competitive nature of FRET and photobleaching is exploited to enable the indirect measurement of FRET via its effect on donor photobleaching lifetimes oil a pixel-by-pixel basis. The method allows enhanced resolution between 10 and 100 Angstrom and represents a sensitive and specific biophysical tool for characterizing the assembly and regulation of GPCR oligomers on the cell surface. (C) 2002 Elsevier Science (USA). All rights reserved.