In-cell detection of conformational sub-states of a GPCR quaternary structure: Modulation of sub-state probability by cognate ligand binding

While the notion that G protein-coupled receptors (GPCRs) associate into homo- and hetero-oligomers has gained more recognition in recent years, a lack of consensus remains among researchers regarding the functional relevance of GPCR oligomerization. A relatively recent technique, Forster resonance energy transfer (FRET) spectrometry, allows for the determination of the oligomeric (or quaternary) structure of proteins in living cells via analysis of efficiency distributions of energy transferred from optically excited fluorescent tags acting as donors of energy to fluorescent tags acting as acceptors of energy and residing within the same oligomer. In this study, we significantly improved the resolution of the FRET-spectrometry approach to detect small differences between the interprotomeric distances among GPCR oligomers with subtle differences in quaternary structures. We then used this approach to study the conformational substates of oligomers of sterile 2 α-factor receptor (Ste2), a class D GPCR found in the yeast Saccharomyces cerevisiae of mating type a. Ste2 has previously been shown to form tetrameric oligomers at relatively low expression levels (between 11 and 140 molecules/μm2) in the absence of its cognate ligand, the α-factor pheromone. The significantly improved FRET spectrometry technique allowed us to detect multiple distinct quaternary conformational substates of Ste2 oligomers, and to assess how the α-factor ligand altered the proportion of such substates. The ability to determine quaternary structure substates of GPCRs provides exquisite means to elucidate functional relevance of GPCR oligomerization.