Monomeric G protein-coupled receptor rhodopsin in solution activates its G protein transducin at the diffusion limit
Monomeric G protein-coupled receptor rhodopsin in solution activates its G protein transducin at the diffusion limit
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DOI：
10.1073/pnas.0701967104
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发表时间：
2007-06-26
影响因子：
11.1
通讯作者：
Heck, Martin
中科院分区：
文献类型：
--
作者：
Ernst, Oliver P.;Gramse, Verena;Heck, Martin
G蛋白偶联受体通过刺激异源三聚体G蛋白（G α β γ）中的核苷酸交换来介导生物信号。已提出受体二聚体作为负责与G α β γ催化相互作用的功能单元。为了研究G蛋白偶联受体单体是否可以激活G α β γ，我们使用视网膜光感受器视紫红质及其同源G蛋白转导素（G（t））来确定视紫红质/G（t）结合的化学计量和G（t）中催化的核苷酸交换速率。在十二烷基麦芽糖苷洗涤剂溶液中制备纯化的视紫红质。视紫红质是单体的结论，从荧光共振能量转移，copurification研究与荧光标记和未标记的视紫红质，尺寸排阻色谱法，和多角度激光散射。在洗脱曲线中发现光活化视紫红质和G（t）之间的1：1复合物，并且在复合物形成时释放一个分子的GDP。对催化视紫红质/G（t）相互作用的速度的分析产生了每秒大约50个G（t）分子和活化视紫红质分子的最大值。在稀释体系中，双分子速率常数接近扩散极限。结果表明，G（t）与活化的视紫红质单体的相互作用足以使G（t）活化。尽管溶液中的活化速率处于物理上可能的极限，但天然膜中的速率仍然高出10倍。这可能归因于G蛋白对膜表面的精确定向，这使得在实际活化步骤之前能够进行快速对接过程。是否对接在膜涉及视紫红质二聚体或寡聚体的形成仍有待阐明。
G protein-coupled receptors mediate biological signals by stimulating nucleotide exchange in heterotrimeric G proteins (G alpha beta gamma). Receptor dimers have been proposed as the functional unit responsible for catalytic interaction with G alpha beta gamma. To investigate whether a G protein-coupled receptor monomer can activate G alpha beta gamma, we used the retinal photoreceptor rhodopsin and its cognate G protein transducin (G(t)) to determine the stoichiometry of rhodopsin/G(t) binding and the rate of catalyzed nucleotide exchange in G(t). Purified rhodopsin was prepared in dodecyl maltoside detergent solution. Rhodopsin was monomeric as concluded from fluorescence resonance energy transfer, copurification studies with fluorescent labeled and unlabeled rhodopsin, size exclusion chromatography, and multiangle laser light scattering. A 1:1 complex between light-activated rhodopsin and G(t) was found in the elution profiles, and one molecule of GDP was released upon complex formation. Analysis of the speed of catalytic rhodopsin/G(t) interaction yielded a maximum of approximate to 50 G(t) molecules per second and molecule of activated rhodopsin. The bimolecular rate constant is close to the diffusion limit in the diluted system. The results show that the interaction of G(t) with an activated rhodopsin monomer is sufficient for fully functional G(t) activation. Although the activation rate in solution is at the physically possible limit, the rate in the native membrane is still 10-fold higher. This is likely attributable to the precise orientation of the G protein to the membrane surface, which enables a fast docking process preceding the actual activation step. Whether docking in membranes involves the formation of rhodopsin dimers or oligomers remains to be elucidated.