title: Helix 8 of the viral chemokine receptor ORF74 directs chemokine binding.
abstract: The constitutively active G-protein-coupled receptor and viral oncogene ORF74, encoded by Kaposi sarcoma-associated herpesvirus (human herpesvirus 8), binds a broad range of chemokines, including CXCL1 (agonist), CXCL8 (neutral ligand), and CXCL10 (inverse agonist). Although chemokines interact with the extracellular N terminus and loops of the receptor, we demonstrate that helix 8 (Hx8) in the intracellular carboxyl tail (C-tail) of ORF74 directs chemokine binding. Partial deletion of the C-tail resulted in a phenotype with reduced constitutive activity but intact regulation by ligands. Complete deletion of the C-tail, including Hx8, resulted in an inactive phenotype that lacks CXCL8 binding sites and has an increased number of binding sites for CXCL10. Similar effects were obtained with the single R7.61(322)W or Q7.62(323)P mutations in Hx8. We propose that the conserved charged or polar side chain at position 7.61 has a specific role in stabilizing the end of transmembrane domain 7 (TM7). Disruption of Hx8 by deletion or mutation distorts an H-bonding network, involving highly conserved amino acids within TM2, TM7, and Hx8, that is crucial for positioning of the TM domains, coupling to Galphaq, and CXCL8 binding. Thus, Hx8 appears to exert a key role in receptor stabilization through the conserved residue R7.61, directing the ligand binding profile of ORF74 and likely also that of other class A G-protein-coupled receptors.
authors.affiliation: Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Dennis
authors.initials: D
authors.lastname: Verzijl
authors.forename: Leonardo
authors.initials: L
authors.lastname: Pardo
authors.forename: Marie
authors.initials: M
authors.lastname: van Dijk
authors.forename: Yvonne K
authors.initials: YK
authors.lastname: Gruijthuijsen
authors.forename: Aldo
authors.initials: A
authors.lastname: Jongejan
authors.forename: Henk
authors.initials: H
authors.lastname: Timmerman
authors.forename: John
authors.initials: J
authors.lastname: Nicholas
authors.forename: Mario
authors.initials: M
authors.lastname: Schwarz
authors.forename: Philip M
authors.initials: PM
authors.lastname: Murphy
authors.forename: Rob
authors.initials: R
authors.lastname: Leurs
authors.forename: Martine J
authors.initials: MJ
authors.lastname: Smit
mesh_terms: D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D018925:Chemokines / Q000378:metabolism*; D002522:Chlorocebus aethiops; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D005786:Gene Expression Regulation; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D019707:Receptors, Chemokine / Q000737:chemistry* / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D018925:Chemokines; C105181:ORF74 protein, Human herpesvirus 8; D019707:Receptors, Chemokine; D014764:Viral Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11