Spatial association of the Cav1.2 calcium channel with α5β1-integrin
Spatial association of the Cav1.2 calcium channel with α5β1-integrin
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DOI：
10.1152/ajpcell.00171.2010
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发表时间：
2011-03-01
影响因子：
5.5
通讯作者：
Davis, Michael J.
中科院分区：
文献类型：
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作者：
Chao, Jun-Tzu;Gui, Peichun;Davis, Michael J.
Chao J，Gui P，Zamponi GW，Davis GE，Davis MJ. Cav1.2钙通道与α（5）β（1）-整联蛋白的空间关联。美国生理学杂志细胞生理学300：C477-C489，2011年。首次发表于2010年12月22日; doi：10.1152/ajpcell.00171.2010. -在表达CaL的大鼠小动脉平滑肌细胞和人胚肾细胞（HEK293-T）中，FN与α（5）β（1）整合素的结合能显著增强CaV1.2通道（Ca-L）电流。利用免疫共沉淀技术，我们发现HEK293-T细胞中Ca-L与α（5）或β（1）整合素的结合是特异性的，并依赖于细胞与FN的粘附。在大鼠小动脉平滑肌中，CaL和α（5）β（1）-整合素之间以及CaL和磷酸化的c-Src之间的共同关联也被FN处理所揭示和增强。使用HEK293-T细胞中异源表达的Ca-L的定点诱变，我们鉴定了这些相互作用所需的CaL的两个区域：1）COOH末端残基Ser（1901）和Tyr（2122），已知分别被蛋白激酶A（PKA）和c-Src磷酸化;和2）靠近COOH末端中间的两个富含脯氨酸的结构域（PRD）。免疫荧光共聚焦成像显示了中等程度的野生型Ca-L与β（1）-整合素在质膜上的共定位。总的来说，我们的结果强烈地表明：1）在FN连接后，CaL与α（5）β（1）-整联蛋白在包括PKA、c-Src和潜在的其它蛋白激酶的大分子复合物中缔合; 2）CaL与α（5）β（1）-整联蛋白缔合需要CaL在Y-2122和/或S-1901处的磷酸化;和3）c-Src通过与位于CaL的II-III接头区和/或COOH末端的PRD结合，介导α（5）β（1）-整联蛋白接合后的电流增强。这些发现为α（5）β（1）-整合素和FN之间的相互作用如何调节CaL进入并因此改变多种类型的可兴奋细胞的生理功能提供了新的证据。
Chao J, Gui P, Zamponi GW, Davis GE, Davis MJ. Spatial association of the Cav1.2 calcium channel with alpha(5)beta(1)- integrin. Am J Physiol Cell Physiol 300: C477-C489, 2011. First published December 22, 2010; doi:10.1152/ajpcell.00171.2010.-Engagement of alpha(5)beta(1)-integrin by fibronectin (FN) acutely enhances Cav1.2 channel (Ca-L) current in rat arteriolar smooth muscle and human embryonic kidney cells (HEK293-T) expressing CaL. Using coimmunoprecipitation strategies, we show that coassociation of Ca-L with alpha(5)- or beta(1)-integrin in HEK293-T cells is specific and depends on cell adhesion to FN. In rat arteriolar smooth muscle, coassociations between CaL and alpha(5)beta(1)-integrin and between CaL and phosphorylated c-Src are also revealed and enhanced by FN treatment. Using site-directed mutagenesis of Ca-L heterologously expressed in HEK293-T cells, we identified two regions of CaL required for these interactions: 1) COOH- terminal residues Ser(1901) and Tyr(2122), known to be phosphorylated by protein kinase A (PKA) and c-Src, respectively; and 2) two proline-rich domains (PRDs) near the middle of the COOH terminus. Immunofluorescence confocal imaging revealed a moderate degree of wild-type Ca-L colocalization with beta(1)- integrin on the plasma membrane. Collectively, our results strongly suggest that 1) upon ligation by FN, CaL associates with alpha(5)beta(1)-integrin in a macromolecular complex including PKA, c-Src, and potentially other protein kinases; 2) phosphorylation of CaL at Y-2122 and/or S-1901 is required for association of CaL with alpha(5)beta(1)-integrin; and 3) c-Src, via binding to PRDs that reside in the II-III linker region and/or the COOH terminus of CaL, mediates current potentiation following alpha(5)beta(1)-integrin engagement. These findings provide new evidence for how interactions between alpha(5)beta(1)-integrin and FN can modulate CaL entry and consequently alter the physiological function of multiple types of excitable cells.