Mechanism of phosphorylation-recognition by visual arrestin and the transition of arrestin into a high affinity binding state
Mechanism of phosphorylation-recognition by visual arrestin and the transition of arrestin into a high affinity binding state
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DOI：
10.1124/mol.51.1.161
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发表时间：
1997-01-01
影响因子：
3.6
通讯作者：
Benovic, JL
中科院分区：
文献类型：
--
作者：
Gurevich, VV;Benovic, JL
Arrestin通过与视紫红质(P-Rh*)的磷酸化光激活形式特异地相互作用，在猝灭光信号转导过程中发挥重要作用。以前的研究表明，牛arrestin中的Arg175直接参与arrestin与视紫红质的磷酸化依赖的结合，似乎是一个磷酸化敏感的触发器。在这项研究中，我们用19种不同的氨基酸取代Arg175，进一步探讨了磷酸化识别的分子机制。我们还评估了磷酸化识别区域内其他几个高度保守的残基(Val170、Leu172、Leu173、IIe174、Val177和Gln178)的突变效果。然后鉴定了所有这些突变体与P-Rh*、光激活视紫红质和缺少羧基末端磷酸化位点的截短视紫质的结合。总体而言，我们的结果表明，arrestin与视紫红质的磷酸化羧基末端结构域的相互作用激活了arrestin中两个相对独立的变化：(A)额外结合位点的动员和(B)视紫红质羧基末端结构域的磷酸化识别区域的亲和力增加。这两种机制共同确保了arrestin对P-Rh*的精致选择性。对在结合位点动员和磷酸化识别中起主要作用的残基的突变使我们能够创造出对P-Rh*和光激活视紫红质具有高亲和力的“结构性活性”(磷酸化非依赖性)arrestin突变体。在175号位置引入负电荷在这方面特别有效。提出了一个详细的磷酸化识别的分子模型。
Arrestin plays an important role in quenching phototransduction via its ability to interact specifically with the phosphorylated light-activated form of the visual receptor rhodopsin (P-Rh*). Previous studies have demonstrated that Arg175 in bovine arrestin is directly involved in the phosphorylation-dependent binding of arrestin to rhodopsin and seems to function as a phosphorylation-sensitive trigger. In this study, we further probed the molecular mechanism of phosphorylation recognition by substituting 19 different amino acids for Arg175. We also assessed the effects of mutagenesis of several other highly conserved residues within the phosphorylation-recognition region (Val170, Leu172, Leu173, IIe174, Val177, and Gln178). The binding of all of these mutants to P-Rh*, light-activated rhodopsin, and truncated rhodopsin, which lacks the carboxyl-terminal phosphorylation sites, was then characterized. Overall, our results suggest that arrestin interaction with the phosphorylated carboxyl-terminal domain of rhodopsin activates two relatively independent changes in arrestin: (a) mobilization of additional binding sites and (b) increased affinity of the phosphorylation-recognition region for the rhodopsin carboxyl-terminal domain. Together, these two mechanisms ensure the exquisite selectivity of arrestin toward P-Rh*. Mutagenesis of residues that play a major role in binding site mobilization and phosphorylation-recognition enabled us to create ''constitutively active'' (phosphorylation-independent) arrestin mutants that have high affinity for both P-Rh* and light-activated rhodopsin. The introduction of a negative charge in position 175 was particularly effective in this respect. A detailed molecular model of phosphorylation-recognition is proposed.