Identification of regions of arrestin that bind to rhodopsin.
Identification of regions of arrestin that bind to rhodopsin.
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鉴定与视紫红质结合的视紫红质抑制蛋白区域。
DOI：
10.1021/bi982643l
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Hargrave,PA
中科院分区：
文献类型：
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作者：
Smith,WC;McDowell,JH;Dugger,DR;Miller,R;Arendt,A;Popp,MP;Hargrave,PA
Arrestin通过结合光活化和磷酸化的视紫红质（R*P）促进光转导失活。然而，结合到R*P的抑制蛋白的具体部分是未知的。在这项研究中，使用两种不同的方法来确定结合视紫红质的arrestin的区域：使用跨越整个arrestin蛋白的合成arrestin肽淘选噬菌体展示的arrestin片段对R*P和cGMP磷酸二酯酶（PDE）活性抑制。噬菌体展示表明结合的主要区域包含在氨基酸90 - 140内。该区域的一部分（残基95 - 140）表达为与谷胱甘肽S-转移酶的融合蛋白，能够与视紫红质结合，而不管受体的活化或磷酸化状态如何。在这个区域内，残基109 - 130的合成肽显示完全抑制arrestin与视紫红质的结合，IC 50为1.1 mM。这种竞争的相对高的IC 50表明分子的这一部分可能只是arrestin和R* 之间结合的几个区域之一。P.在PDE测定中对合成抑制蛋白肽的研究表明，PDE活性的两种最有效的抑制剂是残基111 - 130和101 - 120的肽。这些结果表明，arrestin和R*P之间至少有一个主要结合区域包含在残基109 - 130的区域内。
Arrestin facilitates phototransduction inactivation through binding to photoactivated and phosphorylated rhodopsin (R*P). However, the specific portions of arrestin that bind to R*P are not known. In this study, two different approaches were used to determine the regions of arrestin that bind to rhodopsin: panning of phage-displayed arrestin fragments against R*P and cGMP phosphodiesterase (PDE) activity inhibition using synthetic arrestin peptides spanning the entire arrestin protein. Phage display indicated the predominant region of binding was contained within amino acids 90−140. A portion of this region (residues 95−140) expressed as a fusion protein with glutathioneS-transferase is capable of binding to rhodopsin regardless of the activation or phosphorylation state of the receptor. Within this region, the synthetic peptide of residues 109−130 was shown to completely inhibit the binding of arrestin to rhodopsin with an IC50of 1.1 mM. The relatively high IC50of this competition suggests that this portion of the molecule may be only one of several regions of binding between arrestin and R*P. A survey of synthetic arrestin peptides in the PDE assay indicated that the two most effective inhibitors of PDE activity were peptides of residues 111−130 and 101−120. These results indicate that at least one of the principal regions of binding between arrestin and R*P is contained within the region of residues 109−130.