Arrestin with a single amino acid substitution quenches light-activated rhodopsin in a phosphorylation-independent fashion
Arrestin with a single amino acid substitution quenches light-activated rhodopsin in a phosphorylation-independent fashion
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DOI：
10.1021/bi963110k
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发表时间：
1997-06-10
期刊：
影响因子：
2.9
通讯作者：
Gurevich, VV
中科院分区：
文献类型：
--
作者：
GrayKeller, MP;Detwiler, PB;Gurevich, VV
抑制蛋白是参与G蛋白介导的信号转导终止的调节蛋白超家族的成员。在脊椎动物视杆细胞的光转导级联反应中，视抑制蛋白的结合是由光活化视紫红质（Rh*）C-末端的磷酸化刺激的，视抑制蛋白作为一种典型的G蛋白介导的信号通路。Arrestin对光激活的磷紫质（P-Rh*）具有很高的选择性。先前我们报道了抑制蛋白Arg 175 Gln中的单个氨基酸取代导致抑制蛋白与Rh* 结合的显著增加[Gurevich，V. V.，& Benovic，J. L.等（1995）J.Biol.Chem.270，6010-6016]。在这里，我们证明了一个类似的突变体，arrestin（R175 E），结合光激活视紫红质独立的磷酸化。Arrestin（R175 E）不仅与P-Rh* 和Ph* 具有高亲和力，而且与光活化的截短视紫红质（其中C-末端磷酸化位点已被蛋白水解去除）具有高亲和力。在监测cGMP磷酸二酯酶（PDE）的视紫红质依赖性活化的体外测定中，野生型抑制蛋白仅在存在ATP以支持视紫红质磷酸化时才淬灭PDE响应。相比之下，在不存在ATP的情况下，低至30 nM的arrestin（R175 E）有效地淬灭PDE活化。当Rh* 的寿命不再对PDE活性的时间过程有贡献时，Arrestin（R175 E）没有影响，表明其在视紫红质-转导素相互作用的水平上破坏信号转导。
Arrestins are members of a superfamily of regulatory proteins that participate in the termination of G protein-mediated signal transduction. In the phototransduction cascade of vertebrate rods, which serves as a prototypical G protein-mediated signaling pathway, the binding of visual arrestin is stimulated by phosphorylation of the C-terminus of photoactivated rhodopsin (Rh*). Arrestin is very selective toward light-activated phosphorhodopsin (P-Rh*). Previously we reported that a single amino acid substitution in arrestin, Arg175Gln, results in a dramatic increase in arrestin binding to Rh* [Gurevich, V. V., & Benovic, J. L. (1995) J. Biol. Chem. 270, 6010-6016]. Here we demonstrate that a similar mutant, arrestin(R175E), binds to light-activated rhodopsin independent of phosphorylation. Arrestin(R175E) binds with high affinity not only to P-Rh* and Ph* but also to light-activated truncated rhodopsin in which the C-terminus phosphorylation sites have been proteolytically removed. In an in vitro assay that monitored rhodopsin-dependent activation of cGMP phosphodiesterase (PDE), wild type arrestin quenched PDE response only when ATP was present to support rhodopsin phosphorylation. In contrast, as little as 30 nM arrestin(R175E) effectively quenched PDE activation in the absence of ATP. Arrestin(R175E) had no effect when the lifetime of Rh* no longer contributed to the time course of PDE activity, suggesting that it disrupts signal transduction at the level of rhodopsin-transducin interaction.