Fraile-Ramos Alberto, Kohout Trudy A, Waldhoer Maria, Marsh Mark
Cell Biology Unit, MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, UK.
Traffic. 2003 Apr;4(4):243-53. doi: 10.1034/j.1600-0854.2003.00079.x.
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 micro2-adaptin subunit of the AP-2 adaptor complex. In cells transfected with micro2 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.
抑制蛋白结合磷酸化的G蛋白偶联受体（GPCR），并通过使受体与其同源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蛋白，将GPCR靶向至内吞网格蛋白包被的囊泡。与细胞GPCR不同，人类巨细胞病毒GPCR和趋化因子受体US28具有组成型信号转导活性，并经历组成型内吞作用。为了确定β-抑制蛋白在US28转运中的作用，我们使用了源自β-抑制蛋白基因敲除小鼠的胚胎成纤维细胞。在这些细胞中，转染的β2-肾上腺素能受体和细胞趋化因子受体CCR5的内化受损。相比之下，US28的分布未受影响，并且在正常和基因敲除细胞系中，US28介导的RANTES内化相似。为了研究网格蛋白介导的途径是否参与US28的内吞作用，我们开发了针对AP-2衔接复合体的μ2-衔接蛋白亚基的小干扰RNA。在转染了μ2小干扰RNA的细胞中，转铁蛋白内吞作用受到严重抑制。抗体摄取实验和生化分析表明，US28的内化也受到抑制。这些数据共同表明，US28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。
