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J. Biol. Chem., Vol. 281, Issue 46, 35327-35335, November 17, 2006

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Helix 8 of the Viral Chemokine Receptor ORF74 Directs Chemokine Binding^*

Dennis Verzijl, Leonardo Pardo, Marie van Dijk, Yvonne K. Gruijthuijsen^¶, Aldo Jongejan, Henk Timmerman, John Nicholas^||1, Mario Schwarz^**, Philip M. Murphy^**, Rob Leurs, and Martine J. Smit²

From the Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands, the Laboratorio de Medicina Computacional, Unidad de Bioestadistica, Facultad de Medicina, Universidad Autonoma de Barcelona, 08193 Barcelona, Spain, the ^¶Department of Medical Microbiology, Cardiovascular Research Institute Maastricht, University of Maastricht, P. O. Box 5800, 6202 AZ Maastricht, The Netherlands, the ^||Molecular Virology Laboratories, Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University, Baltimore, Maryland 21231, and the ^**Laboratory of Molecular Immunology, NIAID, National Institutes of Health, Bethesda, Maryland 20892

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 R7.61³²²W or Q7.62³²³P 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 Gq, 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.

Received for publication, July 19, 2006 , and in revised form, September 20, 2006.

^* This work was supported in part by grants from The Netherlands Organization for Scientific Research (NWO Jonge Chemici grant) (to D. V. and R. L.), the Royal Netherlands Academy of Arts and Sciences (KNAW) (to M. J. S.), and the European Community (Grant LSHB-CT-2003-503337) and Ministerio de Educacion y Ciencia (Grant SAF2006-04966) (to L. P.) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by National Institutes of Health Grants CA76445, CA119887, and CA113239.

² To whom correspondence should be addressed. Tel.: 31-20-5987572; Fax: 31-20-5987610; E-mail: MJ.Smit{at}few.vu.nl.

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