Department of Biological Sciences, Middle East Technical University, Universiteler Mah. Dumlupinar Blv. No: 1, 06800 Cankaya, Ankara, Turkey; Department of Microbiology, University of Tennessee, Knoxville, TN 37996, United States.
Department of Biological Sciences, Middle East Technical University, Universiteler Mah. Dumlupinar Blv. No: 1, 06800 Cankaya, Ankara, Turkey.
Biochim Biophys Acta Biomembr. 2017 May;1859(5):698-711. doi: 10.1016/j.bbamem.2017.01.008. Epub 2017 Jan 8.
Dimerization of G protein-coupled receptors (GPCR) may play an important role in maturation, internalization, signaling and/or pharmacology of these receptors. However, the location where dimerization occurs is still under debate. In our study, variants of Ste2p, a yeast mating pheromone GPCR, were tagged with split EGFP (enhanced green fluorescent protein) fragments inserted between transmembrane domain seven and the C-terminus or appended to the C-terminus. Bimolecular Fluorescence Complementation (BiFC) assay was used to determine where receptor dimerization occurred during protein trafficking by monitoring generation of EGFP fluorescence, which occurred upon GPCR dimerization. Our results suggest that these tagged receptors traffic to the membrane as monomers, undergo dimerization or higher ordered oligomerization predominantly on the plasma membrane, and are internalized as dimers/oligomers. This study is the first to provide direct in vivo visualization of GPCR dimerization/oligomerization, during trafficking to and from the plasma membrane.
G 蛋白偶联受体（GPCR）的二聚化可能在这些受体的成熟、内化、信号转导和/或药理学中发挥重要作用。然而，二聚化发生的位置仍存在争议。在我们的研究中，酵母交配信息素 GPCR Ste2p 的变体被标记为插入跨膜域七和 C 末端之间或附加到 C 末端的分裂 EGFP（增强型绿色荧光蛋白）片段。双分子荧光互补（BiFC）测定用于通过监测 EGFP 荧光的产生来确定受体在蛋白质运输过程中何时发生二聚化，这是在 GPCR 二聚化时发生的。我们的结果表明，这些标记的受体作为单体运输到膜上，主要在质膜上发生二聚化或更高阶的寡聚化，并作为二聚体/寡聚体内化。这项研究首次提供了在质膜内外运输过程中 GPCR 二聚化/寡聚化的直接体内可视化。
