Use of fluorescence resonance energy transfer to analyze oligomerization of G-protein-coupled receptors expressed in yeast.
Oligomerization or dimerization of G-protein-coupled receptors (GPCRs) has emerged as an important theme in signal transduction. This concept has recently gained widespread interest due to the application of direct and noninvasive biophysical techniques such as fluorescence resonance energy transfer (FRET), which have shown unequivocally that several types of GPCR can form dimers or oligomers in living cells. Current challenges are to determine which GPCRs can self-associate and/or interact with other GPCRs, to define the molecular principles that govern these specific interactions, and to establish which aspects of GPCR function require oligomerization. Although these questions ultimately must be addressed by using GPCRs expressed endogenously in their native cell types, analysis of GPCR oligomerization in heterologous expression systems will be useful to survey which GPCRs can interact, to conduct structure-function studies, and to identify peptides or small molecules that disrupt GPCR oligomerization and function. Here, we describe methods employing scanning fluorometry to detect FRET between GPCRs tagged with enhanced cyan and yellow fluorescent proteins (CFP and YFP) in living yeast cells. This approach provides a powerful means to analyze oligomerization of a variety of GPCRs that can be expressed in yeast, such as adrenergic, adenosine, C5a, muscarinic acetylcholine, vasopressin, opioid, and somatostatin receptors.
Overton
Mark C
MC
Department of Cell Biology and Physiology, Box 8228, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.
Blumer
Kendall J
KJ
D001426:Bacterial Proteins / Q000235:genetics; D002462:Cell Membrane; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods* / Q000592:standards; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D015003:Yeasts / Q000235:genetics*
D001426:Bacterial Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D019204:GTP-Binding Proteins