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Journal of Virology, March 2004, p. 2460-2471, Vol. 78, No. 5
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.5.2460-2471.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

G{alpha} Protein Selectivity Determinant Specified by a Viral Chemokine Receptor-Conserved Region in the C Tail of the Human Herpesvirus 8 G Protein-Coupled Receptor

Chaoqi Liu, Gordon Sandford, Guo Fei, and John Nicholas*

Molecular Virology Laboratories, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, Maryland 21231

Received 2 June 2003/ Accepted 12 November 2003

The viral G-protein coupled receptor (vGPCR) specified by human herpesvirus 8 (HHV-8) open reading frame 74 (ORF74) is a ligand-independent chemokine receptor that has structural and functional homologues among other characterized gammaherpesviruses and related receptors in the betaherpesviruses. Sequence comparisons of the gammaherpesvirus vGPCRs revealed a highly conserved region in the C tail, just distal to the seventh transmembrane domain. Mutagenesis of the corresponding codons of HHV-8 ORF74 was carried out to provide C-tail-altered proteins for functional analyses. By measuring receptor-activated vascular endothelial growth factor promoter induction and NF-{kappa}B, mitogen-activated protein kinase, and Ca2+ signaling, we found that while some altered receptors showed general signaling deficiencies, others had distinguishable activation profiles, suggestive of selective G{alpha} protein coupling. This was supported by the finding that vGPCR and representative functionally altered variants, vGPCR.8 (R322W) and vGPCR.15 (M325S), were affected differently by inhibitors of G{alpha}i (pertussis toxin), protein kinase C (GF109203X), and phosphatidylinositol 3-kinase (wortmannin). Consistent with the signaling data, [35S]GTP{gamma}S incorporation assays revealed preferential coupling of vGPCR.15 to G{alpha}q and an inability of vGPCR.8 to couple functionally to G{alpha}q. However, both variants, wild-type vGPCR, and a C-tail deletion version of the receptor were equally able to associate physically with G{alpha}q. Combined, our data demonstrate that HHV-8 vGPCR contains discrete sites of G{alpha} interaction and that receptor residues in the proximal region of the cytoplasmic tail are determinants of G{alpha} protein coupling specificity.

* Corresponding author. Mailing address: Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, 1650 Orleans St., Room 309, Baltimore, MD 21231. Phone: (410) 502-6801. Fax: (410) 502-6802. E-mail: nichojo{at}jhmi.edu.

Journal of Virology, March 2004, p. 2460-2471, Vol. 78, No. 5
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.5.2460-2471.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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