Carrillo Juan J, López-Giménez Juan F, Milligan Graeme
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.
Mol Pharmacol. 2004 Nov;66(5):1123-37. doi: 10.1124/mol.104.001586. Epub 2004 Aug 10.
Combinations of coimmunoprecipitation, single-cell fluorescence resonance energy transfer, and cell-surface time-resolved fluorescence resonance energy transfer demonstrated protein-protein interactions and quaternary structure for the alpha(1b)-adrenoceptor. Self-association of transmembrane domain 1 and its interaction with the full-length receptor indicated a symmetrical interface provided by this domain. Lack of effect of mutation of the glycophorin-A dimerization-like region within this helix demonstrated that this did not provide the molecular mechanism. Multiple interactions were observed between the alpha(1b)-adrenoceptor and fragments derived from its sequence. Fragments comprising transmembrane domains 3 and 4 and transmembrane domains 5 and 6, but not transmembrane domain 7, were also able to interact with the full-length receptor. Transmembrane domain 7 failed to interact significantly with any element of the receptor and was not transported to the cell surface after coexpression with the full-length receptor. Symmetrical interactions were also noted between fragments incorporating transmembrane domain 4, but this segment of the receptor failed to interact with transmembrane domains 1 and 2 or transmembrane domains 5 and 6. Time-resolved fluorescence resonance energy transfer studies were also consistent with contributions of transmembrane domains 1 and/or 2 and transmembrane domains 3 and/or 4 to protein-protein interactions within the quaternary structure of the alpha(1b)-adrenoceptor, and with a contribution of transmembrane domains 5 and/or 6. These data are consistent with a complex oligomeric quaternary structure of the alpha(1b)-adrenoceptor in which major, symmetrical interactions may define intradimeric contacts with other contributions, providing interdimer contacts to generate oligomeric complexes akin to those observed for murine rhodopsin. A model derived from this was developed.
免疫共沉淀、单细胞荧光共振能量转移和细胞表面时间分辨荧光共振能量转移相结合的方法，证实了α(1b)-肾上腺素能受体的蛋白质-蛋白质相互作用和四级结构。跨膜结构域1的自缔合及其与全长受体的相互作用表明，该结构域提供了一个对称界面。该螺旋内糖蛋白A二聚化样区域的突变缺乏效应，表明这不是分子机制。观察到α(1b)-肾上腺素能受体与其序列衍生片段之间存在多种相互作用。包含跨膜结构域3和4以及跨膜结构域5和6的片段，但不包括跨膜结构域7，也能够与全长受体相互作用。跨膜结构域7未能与受体的任何元件发生显著相互作用，并且在与全长受体共表达后未转运到细胞表面。在包含跨膜结构域4的片段之间也观察到对称相互作用，但该受体片段未能与跨膜结构域1和2或跨膜结构域5和6相互作用。时间分辨荧光共振能量转移研究也与跨膜结构域1和/或2以及跨膜结构域3和/或4对α(1b)-肾上腺素能受体四级结构内蛋白质-蛋白质相互作用的贡献一致，也与跨膜结构域5和/或6的贡献一致。这些数据与α(1b)-肾上腺素能受体的复杂寡聚四级结构一致，其中主要的对称相互作用可能定义二聚体内的接触，其他贡献提供二聚体间的接触，以产生类似于小鼠视紫红质的寡聚复合物。由此得出了一个模型。
