| Title: | Quaternary structure of the yeast pheromone receptor Ste2 in living cells |
| Author(s): | Stoneman MR; Paprocki JD; Biener G; Yokoi K; Shevade A; Kuchin S; Raicu V; |
| Address: | "Physics Department, University of Wisconsin-Milwaukee, Milwaukee, WI, USA. Electronic address: stonema2@uwm.edu. Physics Department, University of Wisconsin-Milwaukee, Milwaukee, WI, USA. Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI, USA. Physics Department, University of Wisconsin-Milwaukee, Milwaukee, WI, USA; Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI, USA. Electronic address: vraicu@uwm.edu" |
| Journal Title: | Biochim Biophys Acta Biomembr |
| DOI: | 10.1016/j.bbamem.2016.12.008 |
| ISSN/ISBN: | 0005-2736 (Print) 0005-2736 (Linking) |
| Abstract: | "Transmembrane proteins known as G protein-coupled receptors (GPCRs) have been shown to form functional homo- or hetero-oligomeric complexes, although agreement has been slow to emerge on whether homo-oligomerization plays functional roles. Here we introduce a platform to determine the identity and abundance of differing quaternary structures formed by GPCRs in living cells following changes in environmental conditions, such as changes in concentrations. The method capitalizes on the intrinsic capability of FRET spectrometry to extract oligomer geometrical information from distributions of FRET efficiencies (or FRET spectrograms) determined from pixel-level imaging of cells, combined with the ability of the statistical ensemble approaches to FRET to probe the proportion of different quaternary structures (such as dimers, rhombus or parallelogram shaped tetramers, etc.) from averages over entire cells. Our approach revealed that the yeast pheromone receptor Ste2 forms predominantly tetramers at average expression levels of 2 to 25 molecules per pixel (2.8.10(-6) to 3.5.10(-5)molecules/nm(2)), and a mixture of tetramers and octamers at expression levels of 25-100 molecules per pixel (3.5.10(-5) to 1.4.10(-4)molecules/nm(2)). Ste2 is a class D GPCR found in the yeast Saccharomyces cerevisiae of the mating type a, and binds the pheromone alpha-factor secreted by cells of the mating type alpha. Such investigations may inform development of antifungal therapies targeting oligomers of pheromone receptors. The proposed FRET imaging platform may be used to determine the quaternary structure sub-states and stoichiometry of any GPCR and, indeed, any membrane protein in living cells. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova" |
| Keywords: | "Cell Membrane/chemistry/metabolism Fluorescence Resonance Energy Transfer Pheromones/metabolism Protein Multimerization Protein Structure, Quaternary Receptors, G-Protein-Coupled/*chemistry/genetics/metabolism Receptors, Mating Factor/*chemistry/genetics/;" |
| Notes: | "MedlineStoneman, Michael R Paprocki, Joel D Biener, Gabriel Yokoi, Koki Shevade, Aishwarya Kuchin, Sergei Raicu, Valerica eng Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Netherlands 2016/12/21 Biochim Biophys Acta Biomembr. 2017 Sep; 1859(9 Pt A):1456-1464. doi: 10.1016/j.bbamem.2016.12.008. Epub 2016 Dec 16" |
