GPCR-Gα protein precoupling: Interaction between Ste2p, a yeast GPCR, and Gpa1p, its Gα protein, is formed before ligand binding via the Ste2p C-terminal domain and the Gpa1p N-terminal domain

Abstract

G protein coupled receptors bind ligands that initiate intracellular signaling cascades via heterotrimeric G proteins. In this study, involvement of the N-terminal residues of yeast G-alpha (Gpa1p) with the C-terminal residues of a full-length or C-terminally truncated Ste2p were investigated using bioluminescence resonance energy transfer (BRET), a non-radiative energy transfer phenomenon where protein-protein interactions can be quantified between a donor bioluminescent molecule and a suitable acceptor fluorophore. Constitutive and position-dependent BRET signal was observed in the absence of agonist (α-factor). Upon the activation of the receptors with α-factor, no significant change in BRET signal was observed. The location of Ste2p-Gpa1p heterodimer was investigated using confocal fluorescence microscopy and bimolecular fluorescence complementation (BiFC) assay, a technique where two non-fluorescent fragments of a fluorescent protein reassemble in vivo to restore fluorescence property thereby directly reporting a protein-protein interaction. BiFC experiments resulted in a dimerization signal intracellularly during biosynthesis on the endoplasmic reticulum (ER) and on the plasma membrane (PM). The constitutive BRET and BiFC signals observed on ER between Ste2p and Gpa1p in their quiescent and activated states are indicative of pre-coupling between these two proteins. This study is the first to show that the extreme N-terminus of yeast G protein alpha subunit is in close proximity to its receptor. The data suggests a pre-coupled heterodimer prior to receptor activation. The images presented in this study are the first direct in vivo evidence showing the localization of receptor - G protein heterodimers during biosynthesis and before reaching the plasma membrane.

Bioluminescence resonance energy transfer-based biosensors allow monitoring of ligand- and transducer-mediated GPCR conformational changes

“…4). Such ligand-independent conformational changes imposed by the G proteins is consistent with the notion that some receptors may be precoupled to G proteins 31–33 . However, we cannot exclude the possibility that such precoupling is forced by over-expressing the G proteins.…”

Structural Insight into G Protein-Coupled Receptor Signaling Efficacy and Bias between Gs and β-Arrestin

“…The strengthening of the link between the PIF and NPxxY motifs by the L124 M mutation would increase the time spent in active conformations for all agonists, through methionine–aromatic interaction, leading to increased β-arrestin recruitment. Such differences in conformational equilibrium would not affect Gs activation since G protein engagement is faster than β-arrestin recruitment. , The observation that Gs on its own can promote conformational changes associated with activation and several studies indicating the existence of a precoupling between receptors and G proteins , would be compatible with the faster activation rate of Gs compared with β-arrestin. For L124G/S mutants, the main effect would be a reduced stabilization of the inactive states, thus increasing Gs activation at the basal level, while the absence of additional stabilization by the ligands would prevent β-arrestin recruitment.…”

Exploring the free-energy landscape of GPCR activation

“…Although the most straightforward model follows an increase in receptor-G-protein coupling upon agonist binding (e.g., refs. 52 and 53), other studies indicate that GPCRs exist in cells in the form of preformed complexes with their appropriate G-protein partners (54,55). The precoupling concept brings an interesting perspective that can be considered using our energy landscape, if we reassess the chain of events starting from the precoupled states rather than the dissociated ones (as in Figs.…”