| Title: | "GPCR-Galpha protein precoupling: Interaction between Ste2p, a yeast GPCR, and Gpa1p, its Galpha protein, is formed before ligand binding via the Ste2p C-terminal domain and the Gpa1p N-terminal domain" |
| Author(s): | Cevheroglu O; Becker JM; Son CD; |
| Address: | "Department of Microbiology, University of Tennessee, Knoxville, TN 37996-0845, United States; Department of Biological Sciences, Middle East Technical University, Universiteler Mah. Dumlupinar Blv. No: 1, Cankaya, Ankara, 06800, Turkey. Department of Microbiology, University of Tennessee, Knoxville, TN 37996-0845, United States. Department of Biological Sciences, Middle East Technical University, Universiteler Mah. Dumlupinar Blv. No: 1, Cankaya, Ankara, 06800, Turkey. Electronic address: cson@metu.edu.tr" |
| Journal Title: | Biochim Biophys Acta Biomembr |
| DOI: | 10.1016/j.bbamem.2017.09.022 |
| ISSN/ISBN: | 0005-2736 (Print) 0005-2736 (Linking) |
| 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 (alpha-factor). Upon the activation of the receptors with alpha-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: | "Bioluminescence Resonance Energy Transfer Techniques Cell Membrane/chemistry/*metabolism Endoplasmic Reticulum/chemistry/*metabolism GTP-Binding Protein alpha Subunits, Gq-G11/genetics/*metabolism Gene Expression Genes, Reporter Green Fluorescent Proteins;" |
| Notes: | "MedlineCevheroglu, Orkun Becker, Jeffrey M Son, Cagdas D eng GM112496/NH/NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Netherlands 2017/09/30 Biochim Biophys Acta Biomembr. 2017 Dec; 1859(12):2435-2446. doi: 10.1016/j.bbamem.2017.09.022. Epub 2017 Sep 27" |
