Endocytosis of the viral chemokine receptor US28 does not require beta-arrestins but is dependent on the clathrin-mediated pathway
Endocytosis of the viral chemokine receptor US28 does not require beta-arrestins but is dependent on the clathrin-mediated pathway
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DOI：
10.1034/j.1600-0854.2003.00079.x
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发表时间：
2003-04-01
期刊：
影响因子：
4.5
通讯作者：
Marsh, M
中科院分区：
文献类型：
--
作者：
Fraile-Ramos, A;Kohout, TA;Marsh, M
抑制蛋白结合磷酸化的G蛋白偶联受体（GPCR）并通过将受体从其同源G蛋白解偶联来抑制激动剂诱导的信号转导。β-抑制蛋白还作为衔接子，将GPCR靶向内吞网格蛋白包被的囊泡。与细胞GPCR不同，人巨细胞病毒GPCR和趋化因子受体US 28显示组成性信号转导活性并经历组成性内吞作用。为了确定β-抑制蛋白在US 28运输中的作用，我们使用了来源于β-抑制蛋白敲除小鼠的胚胎成纤维细胞。在这些细胞中，转染的β 2-肾上腺素能受体和细胞趋化因子受体CCR 5的内化受损。相比之下，US 28分布不受影响，US 28介导的RANTES内化在正常和敲除细胞系中相似。为了研究网格蛋白介导的途径是否参与US 28内吞作用，我们开发了针对AP-2接头复合物的mu 2-adaptin亚基的小干扰RNA。在用μ 2小干扰RNA转染的细胞中，转铁蛋白内吞作用被严重抑制。抗体喂养实验和生化分析表明，US 28内化也受到抑制。总之，这些数据表明US 28内吞作用通过网格蛋白介导的机制发生，但不依赖于β-抑制蛋白。
Arrestins bind phosphorylated G-protein coupled-receptors (GPCR) and inhibit agonist-induced signal transduction by uncoupling the receptors from their cognate G-proteins. beta-arrestins also act as adaptors that target GPCR to endocytic clathrin-coated vesicles. Unlike cellular GPCRs, the human cytomegalovirus GPCRs and chemokine receptor, US28, shows constitutive signal transduction activity and undergoes constitutive endocytosis. To determine the role of beta-arrestins in US28 trafficking, we used embryonic fibroblasts derived from beta-arrestin knockout mice. In these cells, the internalization of transfected beta2-adrenergic receptor and of the cellular chemokine receptor CCR5 was impaired. By contrast, US28 distribution was unaffected, and US28-mediated RANTES internalization was similar in normal and knockout cell lines. To investigate whether a clathrin-mediated pathway is involved in US28 endocytosis, we developed small interfering RNA against the mu2-adaptin subunit of the AP-2 adaptor complex. In cells transfected with mu2 small interfering RNA transferrin endocytosis was severely inhibited. Antibody-feeding experiments and biochemical analysis showed that US28 internalization was also inhibited. Together, these data indicate that US28 endocytosis occurs via a clathrin-mediated mechanism but is independent of beta-arrestins .