Gehret Austin U, Bajaj Anshika, Naider Fred, Dumont Mark E
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642.
Department of Chemistry, College of Staten Island, City University of New York, New York, New York 10314.
J Biol Chem. 2006 Jul 28;281(30):20698-20714. doi: 10.1074/jbc.M513642200. Epub 2006 May 18.
Oligomerization of G protein-coupled receptors is commonly observed, but the functional significance of oligomerization for this diverse family of receptors remains poorly understood. We used bioluminescence resonance energy transfer (BRET) to examine oligomerization of Ste2p, a G protein-coupled receptor that serves as the receptor for the alpha-mating pheromone in the yeast Saccharomyces cerevisiae, under conditions where the functional effects of oligomerization could be examined. Consistent with previous results from fluorescence resonance energy transfer (Overton, M. C., and Blumer, K. J. (2000) Curr. Biol. 10, 341-344), we detected efficient energy transfer between Renilla luciferase and a modified green fluorescent protein individually fused to truncated alpha-factor receptors lacking the cytoplasmic C-terminal tail. In addition, the low background of the BRET system allowed detection of significant, but less efficient, energy transfer between full-length receptors. The reduced efficiency of energy transfer between full-length receptors does not appear to result from different levels of receptor expression. Instead, attachment of fluorescent reporter proteins to the full-length receptors appears to significantly increase the distance between reporters. Mutations that were previously reported to block dimerization of truncated alpha-factor receptors reduce but do not completely eliminate BRET transfer between receptors. Dominant negative effects of mutant alleles of alpha-factor receptors appear to be mediated by receptor oligomerization since these effects are abrogated by introduction of additional mutations that reduce oligomerization. We find that heterodimers of normal and dominant negative receptors are defective in their ability to signal. Thus, signal transduction by oligomeric receptors appears to be a cooperative process requiring an interaction between functional monomers.
G蛋白偶联受体的寡聚化现象普遍存在，但对于这个多样化受体家族而言，寡聚化的功能意义仍知之甚少。我们利用生物发光共振能量转移（BRET）技术，在能够检测寡聚化功能效应的条件下，研究酿酒酵母中作为α交配信息素受体的G蛋白偶联受体Ste2p的寡聚化情况。与荧光共振能量转移的先前结果一致（Overton, M. C., and Blumer, K. J. (2000) Curr. Biol. 10, 341 - 344），我们检测到分别与缺失细胞质C末端尾巴的截短α因子受体融合的海肾荧光素酶和修饰的绿色荧光蛋白之间存在高效的能量转移。此外，BRET系统的低背景使得能够检测到全长受体之间显著但效率较低的能量转移。全长受体之间能量转移效率的降低似乎并非源于受体表达水平的差异。相反，荧光报告蛋白与全长受体的连接似乎显著增加了报告蛋白之间的距离。先前报道的阻断截短α因子受体二聚化的突变会减少但不会完全消除受体之间的BRET转移。α因子受体突变等位基因的显性负效应似乎是由受体寡聚化介导的，因为引入减少寡聚化的额外突变可消除这些效应。我们发现正常受体与显性负受体的异二聚体在信号传导能力上存在缺陷。因此，寡聚体受体的信号转导似乎是一个需要功能性单体之间相互作用的协同过程。
