We have previously shown that the interaction of Ca2+/calmodulin with the metabotropic glutamate receptor type 7 (mGluR7) promotes the G-protein-mediated inhibition of voltage-sensitive Ca2+ channels (VSCCs) seen upon agonist activation. Here, we performed a yeast two-hybrid screen of a new-born rat brain cDNA library using the cytoplasmic C-terminal tail of mGluR7 as bait and identified macrophage myristoylated alanine-rich c-kinase substrate (MacMARCKS) as a binding protein. The interaction was confirmed in vitro and in vivo by pull-down assays, immunoprecipitation, and colocalization of mGluR7 and MacMARCKS in transfected HEK293 cells and cultured cerebellar granule cells. Binding of MacMARCKS to mGluR7 was antagonized by Ca2+/calmodulin. In neurons, cotransfection of MacMARCKS with mGluR7, but not mGluR7 mutants unable to bind MacMARCKS, reduced the G-protein-mediated tonic inhibition of VSCCs in the absence of mGluR7 agonist. These results suggest that competitive interactions of Ca2+/calmodulin and MacMARCKS with mGluR7 control the tonic inhibition of VSCCs by G-proteins.

译文

:我们之前已经证明,Ca2 /钙调蛋白与7型代谢型谷氨酸受体(mGluR7)的相互作用促进了激动剂激活后G蛋白介导的对电压敏感Ca2通道(VSCC)的抑制。在这里,我们使用mGluR7的胞质C末端尾巴作为诱饵,对新生大鼠大脑cDNA文库进行了酵母双杂交筛选,并鉴定了巨噬细胞肉豆蔻酰化的富含丙氨酸的c激酶底物(MacMARCKS)作为结合蛋白。通过下拉测定,免疫沉淀以及mGluR7和MacMARCKS在转染的HEK293细胞和培养的小脑颗粒细胞中的共定位,在体外和体内证实了这种相互作用。 Ca2 /钙调蛋白可拮抗MacMARCKS与mGluR7的结合。在神经元中,将MacMARCKS与mGluR7共转染,但不能与不能结合MacMARCKS的mGluR7突变体共转染,可在不存在mGluR7激动剂的情况下减少VSCC的G蛋白介导的强直抑制。这些结果表明Ca2 /钙调蛋白和MacMARCKS与mGluR7的竞争性相互作用控制了G蛋白对VSCC的强直抑制。

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