The role of phosphorylation in D1 dopamine receptor desensitization -: Evidence for a novel mechanism of arrestin association
The role of phosphorylation in D1 dopamine receptor desensitization -: Evidence for a novel mechanism of arrestin association
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DOI：
10.1074/jbc.m308281200
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发表时间：
2004-02-27
影响因子：
4.8
通讯作者：
Kim, KM
中科院分区：
文献类型：
--
作者：
Kim, OJ;Gardner, BR;Kim, KM
D-1多巴胺受体的同源性脱敏被认为是通过它们的磷酸化导致抑制蛋白缔合而发生的，抑制蛋白缔合阻断G蛋白偶联。为了鉴定受体磷酸化的相关结构域，并确定这如何导致抑制蛋白缔合，我们创建了一系列突变的D-1受体构建体。在一个突变体中，第3胞质结构域内的所有丝氨酸/苏氨酸残基被改变（3rdTOT）。产生了第二构建体，其中这些丝氨酸中仅三个（丝氨酸256、258和259）被突变（3rd 234）。我们还创建了四个羧基末端的截短突变体（T347、T369、T394和T404）。所有这些构建体在表达和腺苷酸环化酶活化方面与野生型受体相当。相反，第三环突变体表现出减弱激动剂诱导的受体磷酸化，这与受损的脱敏反应。受体羧基末端的连续截短导致激动剂诱导的磷酸化的连续损失。在最严重的截短T347突变体中未观察到磷酸化。令人惊讶的是，所有截短的受体表现出正常的脱敏。受体结构促进抑制蛋白协会的能力进行了评估，使用抑制蛋白-绿色荧光蛋白易位测定和共聚焦荧光显微镜。3rd 234突变体受体诱导arrestin易位的能力受损，而T347突变体与野生型相当。我们的数据表明，在一个模型中，arrestin直接与激活的第三胞质结构域的激动剂依赖的方式，然而，在基础条件下，这是空间阻止的受体的羧基末端。受体活化促进残基的顺序磷酸化，首先在羧基末端内，然后在第3胞质环内，从而解离这些结构域并允许抑制蛋白结合至活化的第3环。因此，受体磷酸化的作用是允许抑制蛋白进入其受体结合结构域，而不是产生抑制蛋白结合位点本身。
Homologous desensitization of D-1 dopamine receptors is thought to occur through their phosphorylation leading to arrestin association which interdicts G protein coupling. In order to identify the relevant domains of receptor phosphorylation, and to determine how this leads to arrestin association, we created a series of mutated D-1 receptor constructs. In one mutant, all of the serine/threonine residues within the 3rd cytoplasmic domain were altered (3rdTOT). A second construct was created in which only three of these serines (serines 256, 258, and 259) were mutated (3rd234). We also created four truncation mutants of the carboxyl terminus (T347, T369, T394, and T404). All of these constructs were comparable with the wild-type receptor with respect to expression and adenylyl cyclase activation. In contrast, both of the 3rd loop mutants exhibited attenuated agonist-induced receptor phosphorylation that was correlated with an impaired desensitization response. Sequential truncation of the carboxyl terminus of the receptor resulted in a sequential loss of agonist-induced phosphorylation. No phosphorylation was observed with the most severely truncated T347 mutant. Surprisingly, all of the truncated receptors exhibited normal desensitization. The ability of the receptor constructs to promote arrestin association was evaluated using arrestin-green fluorescent protein translocation assays and confocal fluorescence microscopy. The 3rd234 mutant receptor was impaired in its ability to induce arrrestin translocation, whereas the T347 mutant was comparable with wild type. Our data suggest a model in which arrestin directly associates with the activated 3rd cytoplasmic domain in an agonist-dependent fashion; however, under basal conditions, this is sterically prevented by the carboxyl terminus of the receptor. Receptor activation promotes the sequential phosphorylation of residues, first within the carboxyl terminus and then the 3rd cytoplasmic loop, thereby dissociating these domains and allowing arrestin to bind to the activated 3rd loop. Thus, the role of receptor phosphorylation is to allow access of arrestin to its receptor binding domain rather than to create an arrestin binding site per se.