In-Cell Detection of Conformational Substates of a G Protein-Coupled Receptor Quaternary Structure: Modulation of Substate Probability by Cognate Ligand Binding

Abstract

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, Förster 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/µm 2 ) 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.

Modulation of adenosine A2a receptor oligomerization by receptor activation and PIP2 interactions

“…Such a shape made branched/closed quaternary structures more energetically favorable. Dynamic and compact oligomeric structures have been reported for the Ste2 receptor by FRET ( Paprocki et al., 2020 ). The coupling of mini Gs further enhanced this shift to more compact configurations in which some of the signaling partners made contacts with one another.…”

“…The adenosine 2a receptor (A2aR) was found to form higher-order oligomers at the plasma membranes ( Glukhova et al., 2017 ; Vidi et al., 2008a ), whereas β 1 adrenergic receptors formed only monomers and dimers ( Calebiro et al., 2013 ). The kinetics of GPCR oligomerization has been reported to be dynamic ( Dijkman et al., 2018 ) and regulated by receptor density ( Calebiro et al., 2013 ) and by ligand ( Möller et al., 2020 ; Paprocki et al., 2020 ). The oligomerization landscape is made more complex by diverse oligomeric structures.…”

Dielectric Spectroscopy Based Detection of Specific and Nonspecific Cellular Mechanisms

“…One type of cell sample contained a pair of plasmids,(pYF1988 and pYF2034), each of which contained a gene encoding the sterile2 α-factor receptor fused in frame to a gene encoding one of two variants of the green fluorescent protein (GFP)—i.e., either GFP2 [ 55 ] (PYF1988) or YFP [ 56 ] (PYF2034). Ste2 is the protein of interest in this study, while the GFP2 and YFP tags serve as markers of the Ste2 in separate fluorescence microscopy-based studies of this receptor [ 20 , 53 , 57 ]. The other type of cell sample, referred to as Ste2Δ throughout the manuscript, contained a pair of plasmids comprising the same DNA as the first plasmid pair except those coding DNA for the Ste2–fluorescent protein complex.…”

“…However, a high degree of uncertainty still clouds the use of these methods for detection of the agonist based GPCR activation. While some investigators have reported large changes to FRET- and BRET-derived signals upon agonist binding to certain GPCRs [ 17 , 18 , 19 , 20 , 21 , 22 ], others have failed to detect any difference in signal after agonist stimulation of other receptors [ 23 , 24 , 25 , 26 , 27 ]. One challenge that such methods face is that agonist-induced conformational changes to the receptors of interest are not necessarily accompanied by significant changes in the position and/or orientation of the fluorescent tags needed to detect the FRET or BRET signal [ 28 ].…”

“…Ste2, a member of the GPCR family, is expressed in yeast cells of the a mating type and binds the pheromone, α-factor, secreted by yeast cells of the α mating type. The binding of α-factor to Ste2 initiates a signal transduction pathway that leads to cell division arrest and mating with an α type yeast cell [ 20 , 52 , 53 ]. Equipped with an applicable electrical model for yeast and a robust data fitting routine, we extracted the dielectric properties of not only the plasma membrane region where the Ste2 receptors are expected to bind the α-factor, but also of the interior of the cell.…”