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

摘要

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. Graphical abstract Display Omitted Highlights ? A model GPCR–G alpha dimerization was studied using Ste2p and Gpa1p. ? Position dependent BRET suggests Gpa1p extreme N-terminus interacts with Ste2p. ? Constitutive BRET and BiFC in quiescent state suggests a precoupled GPCR–G alpha. ? Ste2p-Gpa1p heterodimerize on endoplasmic reticulum before reaching plasma membrane. ? Ste2p-Gpa1p agonist activation proceeds through a molecular rearrangement.