Mutational analysis of predicted intracellular loop domains of human motilin receptor
Mutational analysis of predicted intracellular loop domains of human motilin receptor
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DOI：
10.1152/ajpgi.00244.2007
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发表时间：
2008-02-01
影响因子：
4.5
通讯作者：
Onji, Morikazu
中科院分区：
文献类型：
--
作者：
Tokunaga, Hitoo;Matsuura, Bunzo;Onji, Morikazu
胃动素是胃肠道运动功能的重要内源性调节因子，由I类G蛋白偶联胃动素受体介导。胃动素和红霉素，胃动素受体的两种化学上不同的完全激动剂，已知结合到该受体的不同区域，基于先前的细胞外区域的系统诱变，其分离对这两种药剂的作用。在目前的工作中，我们研究了预测的细胞内环区域的这种受体对胃动素和红霉素刺激活性的影响。我们准备了胃动素受体结构，包括连续删除整个预测的第一，第二和第三胞内环，以及取代关键区域的残基与丙氨酸，苯丙氨酸，或组氨酸。每个构建体在COS细胞中瞬时表达，并表征胃动素和红霉素刺激的细胞内钙反应和胃动素结合。受体残基63 - 66、135 - 137和296 - 301的缺失各自导致对胃动素和红霉素刺激的细胞内钙应答的实质性损失。具有残基Tyr 66、Arg 136和Val 299突变的构建体负责对由两种激动剂刺激的生物活性的负面影响。这些数据表明，不同的化学类别的激动剂，已知与胃动素受体的不同区域相互作用的行动可能会产生一个共同的激活状态的胞质面对这种受体，负责与其G蛋白的相互作用。在预测的胞质面的功能上重要的残基的鉴定提供了强有力的候选人在受体-G蛋白相互作用中发挥作用。
Motilin is an important endogenous regulator of gastrointestinal motor function, mediated by the class I G protein-coupled motilin receptor. Motilin and erythromycin, two chemically distinct full agonists of the motilin receptor, are known to bind to distinct regions of this receptor, based on previous systematic mutagenesis of extracellular regions that dissociated the effects on these two agents. In the present work, we examined the predicted intracellular loop regions of this receptor for effects on motilin-and erythromycin-stimulated activity. We prepared motilin receptor constructs that included sequential deletions throughout the predicted first, second, and third intracellular loops, as well as replacing the residues in key regions with alanine, phenylalanine, or histidine. Each construct was transiently expressed in COS cells and characterized for motilin-and erythromycin-stimulated intracellular calcium responses and for motilin binding. Deletions of receptor residues 63 - 66, 135 - 137, and 296 - 301 each resulted in substantial loss of intracellular calcium responses to stimulation by both motilin and erythromycin. Constructs with mutations of residues Tyr66, Arg136, and Val299 were responsible for the negative impact on biological activity stimulated by both agonists. These data suggest that action by different chemical classes of agonists that are known to interact with distinct regions of the motilin receptor likely yield a common activation state of the cytosolic face of this receptor that is responsible for interaction with its G protein. The identification of functionally important residues in the predicted cytosolic face provides strong candidates for playing roles in receptor-G protein interaction.