Milligan Graeme, Pediani John D, Canals Meritxell, Lopez-Gimenez Juan F
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
Vision Res. 2006 Dec;46(27):4434-41. doi: 10.1016/j.visres.2006.08.007. Epub 2006 Sep 26.
The structural basis of the quaternary organization of rhodopsin has recently been explored and modeled. Because information obtained from studying rhodopsin has frequently been directly applicable to other G protein-coupled receptors we wished to ascertain if dimeric and/or oligomeric forms of the alpha(1b)-adrenoceptor could be observed and if so whether rhodopsin might provide insights into the quaternary structure of this receptor. Co-immunoprecipitation and both conventional and time-resolved fluorescence resonance energy transfer studies demonstrated quaternary structure of the alpha(1b)-adrenoceptor and, in concert with the reconstitution of fragments of this receptor, provided information on the molecular basis of these interactions. Development of three color fluorescence resonance energy transfer (FRET) allowed the imaging of alpha(1b)-adrenoceptor oligomers in single living cells. Mutation of hydrophobic residues in transmembrane domains I and IV of the receptor resulted in marked reduction in three color FRET suggesting an alteration in oligomeric organization and potential similarities with rhodopsin. The mutated alpha(1b)-adrenoceptor was unable to reach the cell surface, did not become terminally N-glycosylated and was unable to signal.
最近，人们对视紫红质四级结构的结构基础进行了探索和建模。由于从对视紫红质的研究中获得的信息常常可以直接应用于其他G蛋白偶联受体，因此我们希望确定是否可以观察到α(1b)-肾上腺素能受体的二聚体和/或寡聚体形式，如果可以，视紫红质是否能为该受体的四级结构提供见解。免疫共沉淀以及传统和时间分辨荧光共振能量转移研究证明了α(1b)-肾上腺素能受体的四级结构，并且与该受体片段的重组相结合，提供了有关这些相互作用分子基础的信息。三色荧光共振能量转移（FRET）技术的发展使得在单个活细胞中对α(1b)-肾上腺素能受体寡聚体进行成像成为可能。受体跨膜结构域I和IV中疏水残基的突变导致三色FRET显著降低，这表明寡聚体组织发生了改变，并且与视紫红质存在潜在相似性。突变的α(1b)-肾上腺素能受体无法到达细胞表面，不会进行终末N-糖基化，并且无法发出信号。
