c-Src regulates clathrin adapter protein 2 interaction with β-arrestin and the angiotensin II type 1 receptor during clathrin-mediated internalization
c-Src regulates clathrin adapter protein 2 interaction with β-arrestin and the angiotensin II type 1 receptor during clathrin-mediated internalization
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DOI：
10.1210/me.2004-0246
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发表时间：
2005-02-01
影响因子：
--
通讯作者：
Laporte, SA
中科院分区：
文献类型：
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作者：
Fessart, D;Simaan, M;Laporte, SA
beta-Arrestins 是参与 G 蛋白偶联受体 (GPCR) 内化和信号传导的多功能接头。它们通过与网格蛋白和网格蛋白接头 2 (AP-2) 复合物结合，将受体靶向网格蛋白包被的凹坑 (CCP)。它们还通过将 c-Src 激酶招募到某些 GPCR 来充当信号转导器。在这里，我们试图确定 c-Src 在内化过程中是否调节 AP-2 向 β-arrestin 和血管紧张素 II (Ang II) 1 型受体 (AT1R) 的募集。我们发现血管平滑肌细胞 (VSMC) 中天然 AT1R 的激动剂刺激会诱导包含 c-Src、β-抑制蛋白和 AP-2 的内源性复合物的形成。使用免疫共沉淀实验和酵母三杂交测定的体外研究表明，c-Src 可以稳定 β-arrestin2 和 AP-2 β 亚基之间的激动剂非依赖性关联，而与 c-Src 的激酶活性无关。然而，尽管c-Src表达在受体刺激后促进AP-2与β-抑制蛋白和AT1R快速解离，但c-Src的激酶失活突变体未能诱导AP-2与激动剂占据的受体解离。因此，通过使用小干扰RNA策略耗尽人胚肾(HEK)293细胞中的c-Src，还检查了c-Src在调节AP-2与受体解离中的结果对AT1R的内化。在 c-Src 耗尽的细胞中进行的实验表明，在 Ang II 刺激后，AT1R 大部分仍与 AP-2 共定位在质膜上，这与观察到的受体内化延迟一致。此外，在 c-Src 耗尽的 HEK 293 细胞和 VSMC 中进行的免疫共沉淀实验显示，AP-2 与激动剂占据的 AT1R 和 β-arrestin 的关联性分别增强。总之，我们的结果支持 c-Src 在网格蛋白介导的受体内化过程中调节 AP-2 与激动剂占据的 AT1R 和 β-arrestin 解离中的作用，并表明 c-Src 激酶在 AT1R 内化中的新功能。
beta-Arrestins are multifunctional adapters involved in the internalization and signaling of G protein-coupled receptors (GPCRs). They target receptors to clathrin-coated pits (CCPs) through binding with clathrin and clathrin adapter 2 (AP-2) complex. They also act as transducers of signaling by recruiting c-Src kinase to certain GPCRs. Here we sought to determine whether c-Src regulates the recruitment of AP-2 to beta-arrestin and the angiotensin II (Ang II) type 1 receptor (AT1R) during internalization. We show that the agonist stimulation of native AT1R in vascular smooth muscle cells (VSMCs) induces the formation of an endogenous complex containing c-Src, beta-arrestins and AP-2. In vitro studies using coimmunoprecipitation experiments and a yeast three-hybrid assay reveal that c-Src stabilizes the agonist-independent association between beta-arrestin2 and the beta-subunit of AP-2 independently of the kinase activity of c-Src. However, although c-Src expression promoted the rapid dissociation of AP-2 from both beta-arrestin and AT1R after receptor stimulation, a kinase-inactive mutant of c-Src failed to induce the dissociation of AP-2 from the agonist-occupied receptor. Thus, the consequence of c-Src in regulating the dissociation of AP-2 from the receptor was also examined on the internalization of AT1R by depleting c-Src in human embryonic kidney (HEK) 293 cells using a small interfering RNA strategy. Experiments in c-Src depleted cells reveal that AT1R remained mostly colocalized with AP-2 at the plasma membrane after Ang II stimulation, consistent with the observed delay in receptor internalization. Moreover, coimmunoprecipitation experiments in c-Src depleted HEK 293 cells and VSMCs showed an increased association of AP-2 to the agonist-occupied AT1R and beta-arrestin, respectively. Together, our results support a role for c-Src in regulating the dissociation of AP-2 from agonist-occupied AT1R and beta-arrestin during the clathrin-mediated internalization of receptors and suggest a novel function for c-Src kinase in the internalization of AT1R.