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

Biochim Biophys Acta Biomembr. 2017 Dec;1859(12):2435-2446. doi: 10.1016/j.bbamem.2017.09.022. Epub 2017 Sep 27.


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.

Keywords: Bimolecular fluorescence complementation (BiFC); Bioluminescence resonance energy transfer (BRET); G protein-coupled receptor (GPCR); GPCR-G protein heterodimerization; Receptor signaling.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bioluminescence Resonance Energy Transfer Techniques
  • Cell Membrane / chemistry
  • Cell Membrane / metabolism*
  • Endoplasmic Reticulum / chemistry
  • Endoplasmic Reticulum / metabolism*
  • GTP-Binding Protein alpha Subunits, Gq-G11 / genetics
  • GTP-Binding Protein alpha Subunits, Gq-G11 / metabolism*
  • Gene Expression
  • Genes, Reporter
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Ligands
  • Mating Factor / genetics
  • Mating Factor / metabolism*
  • Phosphorylation
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Multimerization
  • Receptors, Mating Factor / genetics
  • Receptors, Mating Factor / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Signal Transduction


  • Ligands
  • Receptors, Mating Factor
  • STE2 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • alpha-factor (Saccharomyces cerevisiae)
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Mating Factor
  • GPA1 protein, S cerevisiae
  • GTP-Binding Protein alpha Subunits, Gq-G11