title: Endocytosis of the viral chemokine receptor US28 does not require beta-arrestins but is dependent on the clathrin-mediated pathway.
abstract: 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.
authors.affiliation: Cell Biology Unit, MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, UK.
authors.forename: Alberto
authors.initials: A
authors.lastname: Fraile-Ramos
authors.forename: Trudy A
authors.initials: TA
authors.lastname: Kohout
authors.forename: Maria
authors.initials: M
authors.lastname: Waldhoer
authors.forename: Mark
authors.initials: M
authors.lastname: Marsh
mesh_terms: D000818:Animals; D019390:Arrestins / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D002966:Clathrin / Q000378:metabolism / Q000502:physiology*; D017931:DNA Primers; D004705:Endocytosis; D019204:GTP-Binding Proteins / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D034741:RNA, Small Interfering / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D019707:Receptors, Chemokine / Q000378:metabolism / Q000502:physiology*; D014764:Viral Proteins / Q000378:metabolism / Q000502:physiology*; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D002966:Clathrin; D017931:DNA Primers; D034741:RNA, Small Interfering; D011956:Receptors, Cell Surface; D019707:Receptors, Chemokine; C411191:US28 receptor, Cytomegalovirus; D014764:Viral Proteins; D000071557:beta-Arrestins; D019204:GTP-Binding Proteins