MxA, a member of the dynamin superfamily, interacts with the ankyrin-like repeat domain of TRPC
MxA, a member of the dynamin superfamily, interacts with the ankyrin-like repeat domain of TRPC
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DOI：
10.1074/jbc.m500391200
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发表时间：
2005-05-13
影响因子：
4.8
通讯作者：
Boulay, G
中科院分区：
文献类型：
--
作者：
Lussier, MP;Cayouette, S;Boulay, G
哺乳动物瞬时受体电位经典通道被认为是与非兴奋细胞内钙离子进入活动相关的分子实体。TRPC的氨基酸序列分析显示存在锚定样重复结构域，这是最常见的蛋白质-蛋白质相互作用基序之一。使用酵母双杂交相互作用试验，我们发现，第二个锚样重复结构域的TRPC 6相互作用与MxA，发动蛋白超家族的成员。使用GST下拉和免疫共沉淀试验，我们发现MxA与TRPC 1、-3、-4、-5、-6和-7相互作用。HEK 293 T细胞中MxA的过表达轻微增加了G（q）蛋白偶联受体刺激后的内源性钙内流或毒胡萝卜素消耗。MxA与TRPC 6的共表达增强激动剂诱导的或OAG诱导的钙内流活性。GTP结合缺陷型MxA突变体对OAG诱导的TRPC 6活性只有轻微的增强作用。然而，MxA突变体，可以结合GTP，但缺乏GTP酶活性产生相同的效果MxA对OAG诱导的TRPC 6活性。这些结果表明，MxA特异性地与TRPC的第二个锚样重复结构域相互作用，并表明单体MxA通过需要GTP结合的机制调节TRPC 6的活性。另外的结果显示，由干扰素α处理诱导的MxA内源性表达的增加调节TRPC 6的活性。该研究清楚地确定MxA是一种新的参与Ca 2+信号传导的调节蛋白。
Mammalian transient receptor potential canonical channels have been proposed as the molecular entities associated with calcium entry activity in nonexcitable cells. Amino acid sequence analyses of TRPCs revealed the presence of ankyrin-like repeat domains, one of the most common protein-protein interaction motifs. Using a yeast two-hybrid interaction assay, we found that the second ankyrin-like repeat domain of TRPC6 interacted with MxA, a member of the dynamin superfamily. Using a GST pull-down and co-immunoprecipitation assay, we showed that MxA interacted with TRPC1, -3, -4, -5, -6, and -7. Overexpression of MxA in HEK293T cells slightly increased endogenous calcium entry subsequent to stimulation of G(q) protein-coupled receptors or store depletion by thapsigargin. Co-expression of MxA with TRPC6 enhanced agonist-induced or OAG-induced calcium entry activity. GTP binding-defective MxA mutants had only a minor potentiating effect on OAG-induced TRPC6 activity. However, a MxA mutant that could bind GTP but that lacked GTPase activity produced the same effect as MxA on OAG-induced TRPC6 activity. These results indicated that MxA interacted specifically with the second ankyrin-like repeat domain of TRPCs and suggested that monomeric MxA regulated the activity of TRPC6 by a mechanism requiring GTP binding. Additional results showed that an increase in the endogenous expression of MxA, induced by a treatment with interferon alpha, regulated the activity of TRPC6. The study clearly identified MxA as a new regulatory protein involved in Ca2+ signaling.