title: The carboxyl terminus of human cytomegalovirus-encoded 7 transmembrane receptor US28 camouflages agonism by mediating constitutive endocytosis.
abstract: US28 is one of four 7 transmembrane (7TM) chemokine receptors encoded by human cytomegalovirus and has been shown to both signal and endocytose in a ligand-independent, constitutively active manner. Here we show that the constitutive activity and constitutive endocytosis properties of US28 are separable entities in this viral chemokine receptor. We generated chimeric and mutant US28 proteins that were altered in either their constitutive endocytic (US28 Delta 300, US28 Delta 317, US28-NK1-ctail, and US28-ORF74-ctail) or signaling properties (US28R129A). By using this series of mutants, we show that the cytoplasmic tail domain of US28 per se regulates receptor endocytosis, independent of the signaling ability of the core domain of US28. The constitutive endocytic property of the US28 c-tail was transposable to other 7TM receptors, the herpes virus 8-encoded ORF74 and the tachykinin NK1 receptor (ORF74-US28-ctail and NK1-US28-ctail). Deletion of the US28 C terminus resulted in reduced constitutive endocytosis and consequently enhanced signaling capacity of all receptors tested as assessed by inositol phosphate turnover, NF-kappa B, and cAMP-responsive element-binding protein transcription assays. We further show that the constitutive endocytic property of US28 affects the action of its chemokine ligand fractalkine/CX3CL1 and show that in the absence of the US28 C terminus, fractalkine/CX3CL1 acts as an agonist on US28. This demonstrates for the first time that the endocytic properties of a 7TM receptor can camouflage the agonist properties of a ligand.
authors.affiliation: Laboratory for Molecular Pharmacology, Panum Institute, University of Copenhagen, Copenhagen DK-2200, Denmark. mariaw@egcrc.net
authors.forename: Maria
authors.initials: M
authors.lastname: Waldhoer
authors.forename: Paola
authors.initials: P
authors.lastname: Casarosa
authors.forename: Mette M
authors.initials: MM
authors.lastname: Rosenkilde
authors.forename: Martine J
authors.initials: MJ
authors.lastname: Smit
authors.forename: Rob
authors.initials: R
authors.lastname: Leurs
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Whistler
authors.forename: Thue W
authors.initials: TW
authors.lastname: Schwartz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D054428:Chemokine CX3CL1; D020523:Chemokines, CX3C / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics; D003593:Cytoplasm / Q000737:chemistry; D004705:Endocytosis / Q000502:physiology*; D017353:Gene Deletion; D015870:Gene Expression; D007295:Inositol Phosphates / Q000378:metabolism; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000235:genetics; D011869:Radioligand Assay; D019707:Receptors, Chemokine / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D018040:Receptors, Neurokinin-1 / Q000235:genetics; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
chemical_list: C515538:CX3CL1 protein, human; D054428:Chemokine CX3CL1; D020523:Chemokines, CX3C; D017362:Cyclic AMP Response Element-Binding Protein; D007295:Inositol Phosphates; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D016328:NF-kappa B; C105181:ORF74 protein, Human herpesvirus 8; D019707:Receptors, Chemokine; D018040:Receptors, Neurokinin-1; D011993:Recombinant Fusion Proteins; C411191:US28 receptor, Cytomegalovirus; D014764:Viral Proteins