Rosenzweig Derek H, Nair K Saidas, Levay Konstantin, Peshenko Igor V, Crabb John W, Dizhoor Alexander M, Slepak Vladlen Z
Department of Molecular and Cellular Pharmacology and Neuroscience Program University of Miami Miller School of Medicine, Miami, FL 33136, USA.
Biochem J. 2009 Feb 1;417(3):803-12. doi: 10.1042/BJ20081513.
Vertebrate phototransduction is mediated by cGMP, which is generated by retGC (retinal guanylate cyclase) and degraded by cGMP phosphodiesterase. Light stimulates cGMP hydrolysis via the G-protein transducin, which directly binds to and activates phosphodiesterase. Bright light also causes relocalization of transducin from the OS (outer segments) of the rod cells to the inner compartments. In the present study, we show experimental evidence for a previously unknown interaction between G(alphat) (the transducin alpha subunit) and retGC. G(alphat) co-immunoprecipitates with retGC from the retina or from co-transfected COS-7 cells. The retGC-G(alphat) complex is also present in cones. The interaction also occurs in mice lacking RGS9 (regulator of G-protein signalling 9), a protein previously shown to associate with both G(alphat) and retGC. The G(alphat)-retGC interaction is mediated primarily by the kinase homology domain of retGC, which binds GDP-bound G(alphat) stronger than the GTP[S] (GTPgammaS; guanosine 5'-[gamma-thio]triphosphate) form. Neither G(alphat) nor G(betagamma) affect retGC-mediated cGMP synthesis, regardless of the presence of GCAP (guanylate cyclase activating protein) and Ca2+. The rate of light-dependent transducin redistribution from the OS to the inner segments is markedly accelerated in the retGC-1-knockout mice, while the migration of transducin to the OS after the onset of darkness is delayed. Supplementation of permeabilized photoreceptors with cGMP does not affect transducin translocation. Taken together, these results suggest that the protein-protein interaction between G(alphat) and retGC represents a novel mechanism regulating light-dependent translocation of transducin in rod photoreceptors.
脊椎动物的光转导由cGMP介导，cGMP由视网膜鸟苷酸环化酶（retGC）产生并被cGMP磷酸二酯酶降解。光通过G蛋白转导素刺激cGMP水解，转导素直接结合并激活磷酸二酯酶。强光还会导致转导素从视杆细胞的外段（OS）重新定位到内部隔室。在本研究中，我们展示了G（αt）（转导素α亚基）与retGC之间以前未知相互作用的实验证据。G（αt）与retGC从视网膜或共转染的COS-7细胞中共同免疫沉淀。retGC-G（αt）复合物也存在于视锥细胞中。这种相互作用在缺乏RGS9（G蛋白信号调节剂9）的小鼠中也会发生，RGS9是一种先前显示与G（αt）和retGC都相关的蛋白质。G（αt）-retGC相互作用主要由retGC的激酶同源结构域介导，该结构域与结合GDP的G（αt）的结合力比GTP[S]（GTPγS；鸟苷5'-[γ-硫代]三磷酸）形式更强。无论是否存在鸟苷酸环化酶激活蛋白（GCAP）和Ca2+，G（αt）和G（βγ）都不会影响retGC介导的cGMP合成。在retGC-1基因敲除小鼠中，光依赖性转导素从OS重新分布到内段的速率明显加快，而在黑暗开始后转导素向OS的迁移则延迟。用cGMP补充通透化的光感受器不会影响转导素的易位。综上所述，这些结果表明G（αt）和retGC之间的蛋白质-蛋白质相互作用代表了一种调节视杆光感受器中转导素光依赖性易位的新机制。
