Surface expression and endocytosis of the human cytomegalovirus-encoded chemokine receptor US28 is regulated by agonist-independent phosphorylation.
Human cytomegalovirus encodes the G protein-coupled chemokine receptor homologue US28 that binds several CC chemokines and sequesters extracellular chemokines from the environment of infected cells. Mechanistically, it has been shown that US28 undergoes rapid constitutive receptor endocytosis and recycling. Monoclonal antibodies were raised that allowed the characterization of a ligand-independent phosphorylation and low surface expression of the US28 receptor in transiently transfected HEK293A cells. Phosphoamino acid analysis defined C-terminal serine and threonine residues as phospho-acceptor sites for constitutive receptor phosphorylation. Coexpression of G protein-coupled receptor kinase-2 and US28 enhanced ligand-independent receptor phosphorylation. C-terminal serine to alanine mutagenesis of US28 resulted in a decreased phosphorylation rate that correlated with enhanced surface expression. Maximal surface expression was detected when all C-terminal serines were substituted. Exchange of all C-terminal serines also significantly reduced receptor endocytosis. Thus, constitutive US28 phosphorylation regulates receptor endocytosis and receptor surface display and may thereby provide a pathogenic mechanism for a potential decoy function of the virally encoded receptor.
Mokros
Thilo
T
Max-Delbrück-Center for Molecular Medicine, Department of Tumorgenetics and Immunogenetics, Berlin, Germany.
Rehm
Armin
A
Droese
Jana
J
Oppermann
Martin
M
Lipp
Martin
M
Höpken
Uta E
UE
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D018946:Chemokine CCL5 / Q000378:metabolism; D003587:Cytomegalovirus / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology / Q000821:virology; D017930:Genes, Reporter; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D019707:Receptors, Chemokine / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D018946:Chemokine CCL5; D004791:Enzyme Inhibitors; D016328:NF-kappa B; D019707:Receptors, Chemokine; D011993:Recombinant Fusion Proteins; C411191:US28 receptor, Cytomegalovirus; D014764:Viral Proteins