The N terminus of phosducin is involved in binding of beta gamma subunits of G protein.
The N terminus of phosducin is involved in binding of beta gamma subunits of G protein.
复制标题
磷酸蛋白的 N 末端参与 G 蛋白 β γ 亚基的结合。
DOI：
10.1073/pnas.92.6.2086
复制
发表时间：
1995
影响因子：
11.1
通讯作者：
Simon,MI
中科院分区：
文献类型：
--
作者：
Xu,J;Wu,D;Slepak,VZ;Simon,MI
光导素是一种可溶性磷蛋白，存在于视网膜感光细胞和松果体中。它与鸟嘌呤核苷酸结合蛋白（G蛋白）的β - γ亚基（G β - γ）结合，并可能调节G蛋白的功能。在本研究中，表征了phoducin特定区域结合G β γ的能力。在牛光导素中制备了一系列缺失突变体。在共转染试验中测试了它们抑制G β γ介导的磷脂酶C β 2亚型激活的能力。光导素n端过表达有抑制作用，c端过表达无抑制作用。抑制作用需要前63个氨基酸残基。在第29位残基上，一个保守的11个氨基酸序列中的色氨酸与缬氨酸的替换严重损害了光导素的抑制功能。在大肠杆菌中表达谷胱甘肽s -转移酶-光导蛋白融合蛋白，研究光导蛋白- g β - γ在体外的相互作用。n端63个氨基酸片段能够与G β结合。相反，c端一半不能与G结合。替代突变体很少或没有结合。此外，利用表面等离子体共振技术直接测量了G β γ与光导素片段之间的相互作用，证实了63个氨基酸片段的结合活性和色氨酸残基的重要性。
Phosducin is a soluble phosphoprotein found in retinal photoreceptor cells and in the pineal gland. It binds to the beta gamma subunits of guanine nucleotide-binding proteins (G proteins) (G beta gamma) and may regulate G-protein function. In this study, the ability of specific regions of phosducin to bind G beta gamma was characterized. A series of deletion mutants were made in bovine phosducin. They were tested in cotransfection assays for their ability to inhibit G beta gamma-mediated phospholipase C beta 2 isoform activation. Overexpression of the N-terminal half of phosducin showed inhibition, whereas overexpression of the C-terminal half did not. The first 63 amino acid residues were required for inhibition. A tryptophan-to-valine substitution at residue 29, which is part of a well conserved 11-amino acid sequence, severely impaired phosducin inhibitory function. Glutathione S-transferase-phosducin fusion proteins were expressed in Escherichia coli to study phosducin-G beta gamma interaction in vitro. The N-terminal 63-amino acid fragment was able to bind to G beta gamma. In contrast, the C-terminal half failed to bind to G beta gamma. The substitution mutants showed little or no binding. Furthermore, direct measurements of interaction between G beta gamma and fragments of phosducin, using surface plasmon resonance technology, confirmed the assignment of binding activity to the 63-amino acid fragment and the importance of the tryptophan residue.