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Selective Elimination of High Constitutive Activity or Chemokine Binding in the Human Herpesvirus 8 Encoded Seven Transmembrane Oncogene ORF74

2000; Elsevier BV; Volume: 275; Issue: 34 Linguagem: Inglês

10.1074/jbc.m003800200

ISSN

1083-351X

Autores

Mette M. Rosenkilde, Thomas N. Kledal, Peter Johannes Holst, Thue W. Schwartz,

Tópico(s)

Herpesvirus Infections and Treatments

Resumo

Open reading frame 74 (ORF74) encoded by human herpesvirus 8 is a highly constitutively active seven transmembrane (7TM) receptor stimulated by angiogenic chemokines,e.g. growth-related oncogene-α, and inhibited by angiostatic chemokines e.g. interferon-γ-inducible protein. Transgenic mice expressing ORF74 under control of the CD2 promoter develop highly vascularized Kaposi's sarcoma-like tumors. Through targeted mutagenesis we here create three distinct phenotypes of ORF74: a receptor with normal, high constitutive signaling through the phospholipase C pathway but deprived of binding and action of chemokines obtained through deletion of 22 amino acids from the N-terminal extension; an ORF74 with high constitutive activity but with selective elimination of stimulatory regulation by angiogenic chemokines obtained through substitution of basic residues at the extracellular ends of TM-V or TM-VI; and an ORF74 lacking constitutive activity but with preserved ability to be stimulated by agonist chemokines obtained through introduction of an Asp residue on the hydrophobic, presumed membrane-exposed face of TM-II. It is concluded that careful molecular dissection can selectively eliminate either agonist or inverse agonist modulation as well as high constitutive activity of the virally encoded oncogene ORF74 and that these mutant forms presumably can be used in transgenic animals to identify the molecular mechanism of its transforming activity. Open reading frame 74 (ORF74) encoded by human herpesvirus 8 is a highly constitutively active seven transmembrane (7TM) receptor stimulated by angiogenic chemokines,e.g. growth-related oncogene-α, and inhibited by angiostatic chemokines e.g. interferon-γ-inducible protein. Transgenic mice expressing ORF74 under control of the CD2 promoter develop highly vascularized Kaposi's sarcoma-like tumors. Through targeted mutagenesis we here create three distinct phenotypes of ORF74: a receptor with normal, high constitutive signaling through the phospholipase C pathway but deprived of binding and action of chemokines obtained through deletion of 22 amino acids from the N-terminal extension; an ORF74 with high constitutive activity but with selective elimination of stimulatory regulation by angiogenic chemokines obtained through substitution of basic residues at the extracellular ends of TM-V or TM-VI; and an ORF74 lacking constitutive activity but with preserved ability to be stimulated by agonist chemokines obtained through introduction of an Asp residue on the hydrophobic, presumed membrane-exposed face of TM-II. It is concluded that careful molecular dissection can selectively eliminate either agonist or inverse agonist modulation as well as high constitutive activity of the virally encoded oncogene ORF74 and that these mutant forms presumably can be used in transgenic animals to identify the molecular mechanism of its transforming activity. seven transmembrane human herpesvirus 8 growth related oncogene interferon-γ-inducible protein interleukin phosphatidylinositol open reading frame viral macrophage inflammatory protein Chemokines are chemotactic cytokines that regulate immunological processes through interaction with 7TM1 G-protein-coupled receptors expressed mainly on leukocytes. For example, during inflammation chemokines secure appropriate cell recruitment. Chemokines are also involved in tissue house-holding processes such as angiogenesis (2Horuk R. Nature. 1998; 393: 524-525Crossref PubMed Scopus (51) Google Scholar, 3Tachibana K. Hirota S. Iizasa H. Yoshida H. Kawabata K. Kataoka Y. Kitamura Y. Matsushima K. Yoshida N. Hishikawa S. Kishimoto T. Nagasawa T. Nature. 1998; 393: 591-595Crossref PubMed Scopus (1323) Google Scholar). Genes coding for homologs of mammalian chemokine and chemokine receptors have been found in a number of herpes- and poxviruses (4Wells T.N.C. Schwartz T.W. Curr. Opin. Biotechnol. 1997; 8: 741-748Crossref PubMed Scopus (33) Google Scholar, 5Dairaghi D.J. Greaves D.R. Schall T.J. Semin. Virol. 1998; 8: 377-385Crossref Scopus (28) Google Scholar, 6Lalani A.S. McFadden G. Cytokine Growth Factor Rev. 1999; 10: 219-233Crossref PubMed Scopus (50) Google Scholar, 7Ahuja S.K. Murphy P.M. Herbert C.A. Chemokines in Disease. Humana Press Inc., Totowa, NJ1999Google Scholar). These molecules have presumably been obtained by the virus through an ancient act of molecular piracy and are structurally optimized for a particular pharmacological phenotype of benefit to the virus. The proposed functional properties of virally encoded chemokines are multiple. Some act as chemokine antagonists, for example vMIP-II from HHV-8 (8Kledal T.N. Rosenkilde M.M. Coulin F. Simmons G. Johnsen A.H. Alouani S. Power C.A. Luttichau H.R. Gerstoft J. Clapham P.R. Clark L.I. Wells T.C. Schwartz T.W. Science. 1997; 277: 1656-1659Crossref PubMed Scopus (427) Google Scholar, 9Lüttichau H.R. Stine J. Boesen T.P. Johnsen A.H. Chantry D. Gerstoft J. Schwartz T.W. J. Exp. Med. 2000; 191: 171-180Crossref PubMed Scopus (118) Google Scholar) (HHV-8 is also known as Kaposi's sarcoma-associated herpesvirus) and MC148 from Molluscum contagiosum (9Lüttichau H.R. Stine J. Boesen T.P. Johnsen A.H. Chantry D. Gerstoft J. Schwartz T.W. J. Exp. Med. 2000; 191: 171-180Crossref PubMed Scopus (118) Google Scholar, 10Damon I. Murphy P.M. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6403-6407Crossref PubMed Scopus (105) Google Scholar), and some act as agonists, for example UL146 from human cytomegalovirus (11Penfold M.E.T. Dairaghi D.J. Duke G.M. Saederup N. Mocarski E.S. Kemble G.W. Schall T.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9839-9844Crossref PubMed Scopus (306) Google Scholar) and vMIP-II (12Sozzani S. Luini W. Bianchi G. Allavena P. Wells T.N.C. Napolitano M. Bernardini G. Vecchi A. D'Ambrosio D. Mazzeo D. Sinigaglia F. Santoni A. Maggi E. Romagnani S. Mantovani A. Blood. 1998; 92: 4036-4039Crossref PubMed Google Scholar). In contrast to the viral chemokines, the function of the virally encoded chemokine receptors is not that clear yet. In general these receptors are not required for viral replication in vitro(13Field B.N. Fields Virology. Lippincott-Raven Puplishers, Hagerstown, MD1995Google Scholar). However gene deletion experiments in both mouse and rat cytomegalovirus have shown that, for example the UL33 receptor is essential for targeting and/or replication of the virus in salivary glands (14Davis-Poynter N.J. Lynch D.M. Vally H. Shellam G.R. Rawlinson W.D. Barrell B.G. Farrell H.E. J. Virol. 1997; 71: 1521-1529Crossref PubMed Google Scholar). Several γ2-herpesviruses including HHV-8 (15Chang Y. Cesarman E. Pessin M.S. Lee F. Culpepper J. Knowles D.M. Moore P.S. Science. 1994; 266: 1865-1869Crossref PubMed Scopus (4983) Google Scholar), herpesvirus Saimirii (16Ahuja S.K. Murphy P.M. J. Biol. Chem. 1993; 268: 20691-20694Abstract Full Text PDF PubMed Google Scholar), equine herpesvirus 2 (17Telford E.A. Watson M.S. Aird H.C. Perry J. Davison A.J. J. Mol. Biol. 1995; 249: 520-528Crossref PubMed Scopus (184) Google Scholar), and the murine γ-herpesvirus 68 (18MacDonald M.R. Li X.-Y. Virgin H.W., IV J. Virol. 1997; 71: 1671-1678Crossref PubMed Google Scholar) encode homolog versions of a CXC chemokine receptor with highest homology to CXCR2 among mammalian chemokine receptors. In HHV-8 the receptor is known as ORF74, but it is also frequently referred to as Kaposi's sarcoma-associated herpesvirus-G-protein-coupled receptor (Fig. 1). A prominent pharmacologic feature of ORF74 from HHV-8 is its high degree of constitutive, ligand-independent signaling through the phospholipase C (19Arvanitakis L. Geras-Raaka E. Varma A. Gershengorn M.C. Cesarman E. Nature. 1997; 385: 347-350Crossref PubMed Scopus (575) Google Scholar, 20Rosenkilde M.M. Kledal T.N. Bräuner-Osborne H. Schwartz T.W. J. Biol. Chem. 1999; 274: 956-961Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar) as well as the c-Jun N-terminal kinase and the p38 mitogen-activated protein kinase pathways (21Bais C. Santomasso B. Coso O. Arvanitakis L. Raaka E.G. Gutkind J.S. Asch A.S. Cesarman E. Gerhengorn M.C. Mesri E.A. Nature. 1998; 391: 86-89Crossref PubMed Scopus (749) Google Scholar). Furthermore ORF74 has angiogenic properties and its signaling is closely coupled to production and secretion of vascular endothelial growth factor and to cellular transformation and formation of highly vascularized tumors in SCID mice (21Bais C. Santomasso B. Coso O. Arvanitakis L. Raaka E.G. Gutkind J.S. Asch A.S. Cesarman E. Gerhengorn M.C. Mesri E.A. Nature. 1998; 391: 86-89Crossref PubMed Scopus (749) Google Scholar). In humans, ORF74 is expressed in Kaposi's sarcoma lesions (15Chang Y. Cesarman E. Pessin M.S. Lee F. Culpepper J. Knowles D.M. Moore P.S. Science. 1994; 266: 1865-1869Crossref PubMed Scopus (4983) Google Scholar) and body cavity-associated lymphomas (22Cesarman E. Nador R.G. Bai F. Bohenzky R.A. Russo J.J. Moore P.S. Chang Y. Knowles D.M. J. Virol. 1996; 70: 8218-8223Crossref PubMed Google Scholar) and has been proposed to be causatively involved in these malignancies (21Bais C. Santomasso B. Coso O. Arvanitakis L. Raaka E.G. Gutkind J.S. Asch A.S. Cesarman E. Gerhengorn M.C. Mesri E.A. Nature. 1998; 391: 86-89Crossref PubMed Scopus (749) Google Scholar). Recently, transgenic mice expressing the ORF74 receptor under control of the CD2 promoter have been reported to develop highly vascularized Kaposi's sarcoma-like tumors (1Yang Y.T. Chen S.C. Leach M.W. Manfra D. Homey B. Wiekowski M. Sullivan L. Jenh C.H. Narula S.K. Chensue S.W. Lira S.A. J. Exp. Med. 2000; 191: 445-454Crossref PubMed Scopus (358) Google Scholar), thus supporting the oncogenic potential. ORF74 from HHV-8 binds various human CXC chemokines (19Arvanitakis L. Geras-Raaka E. Varma A. Gershengorn M.C. Cesarman E. Nature. 1997; 385: 347-350Crossref PubMed Scopus (575) Google Scholar, 20Rosenkilde M.M. Kledal T.N. Bräuner-Osborne H. Schwartz T.W. J. Biol. Chem. 1999; 274: 956-961Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). The properties of these chemokines on ORF74 signaling cover the whole pharmacological spectrum: GROα, GROβ and GROγ are agonists, IP-10, stromal cell-derived factor-1α, granulocyte colony stimulating factor-2, and vMIP-II are inverse agonists, whereas the inflammatory CXC chemokines IL-8, neutrophil-activating peptide-2, and epithelial cell-derived activating peptide-78 are neutral ligands, which despite high affinity binding do not affect signaling of the receptor (20Rosenkilde M.M. Kledal T.N. Bräuner-Osborne H. Schwartz T.W. J. Biol. Chem. 1999; 274: 956-961Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Interestingly, the chemokines, which act as agonists on ORF74, are normally angiogenic chemokines in the host, whereas the chemokines that act as inverse agonists are normally angiostatic or angiomodulatory messengers (23Moore B.B. Arenberg D.A. Strieter R.M. Trends Cardiovasc. Sci. 1998; 8: 51-58Crossref PubMed Scopus (13) Google Scholar). Constitutive activity has been described in many 7TM receptors, for instance the adrenergic (24Chidiac P. Hebert T.E. Valiquette M. Dennis M. Bouvier M. Mol. Pharmacol. 1994; 45: 490-499PubMed Google Scholar, 25Samama P.S. Cotecchia T. Costa T. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 4625-4636Abstract Full Text PDF PubMed Google Scholar, 26Scheer A. Fanelli F. Costa T. Cotecchia S. EMBO J. 1996; 15: 3566-3578Crossref PubMed Scopus (361) Google Scholar, 27Scheer A. Fanelli F. Costa T. Benedetti P. Cotecchia S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 808-813Crossref PubMed Scopus (200) Google Scholar), the angiotensin and bradykinin (28Balmforth A.J. Lee A.J. Warburton P. Donelly D. Ball S.G. J. Biol. Chem. 1997; 272: 4245-4251Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 29Marie J. Kock C. Pruneau D. Paquet J.L. Groblewski T. Larguier R. Lombard C. Deslauriers B. Maigret B. Bonnafous J. Mol. Pharmacol. 1999; 55: 92-101Crossref PubMed Scopus (58) Google Scholar), and the glucagon receptors (30Hjorth S.A. Ørskov C. Schwartz T.W. Mol. Endocrinol. 1998; 12: 78-86Crossref PubMed Scopus (58) Google Scholar). The present study is aimed at trying to characterize, in the ORF74 receptor from HHV-8, the structural basis for its broad spectrum binding profile of chemokines as well as the structural basis for its high constitutive activity based on knowledge on ligand binding and signaling properties in other 7TM receptors. Thereby, we created specific ORF74 mutants in which certain elements of the pharmacological repertoire have been selectively eliminated; to exploit these mutants in future transgenic studies to identify the molecular mechanism that HHV-8 has exploited in ORF74 to precipitate the different clinical features of Kaposi's sarcoma and other HHV-8-related malignancies. The human chemokines were purchased from R&D systems (epithelial cell-derived activating peptide-78, GROβ, and GROγ) or kindly provided by Kuldeep Neote, Pfizer (IP-10, granulocyte colony stimulating factor-2, and GROα), Timothy N. C. Wells, Serono Pharmaceutical Research Group, Ares-Serono, Geneva (neutrophil-activating peptide-2 and vMIP-II), Mikael Luther, Glaxo Wellcome (Met stromal cell-derived factor-1α), or Thomas P. Boesen at this laboratory (IL-8). The ORF74 (GenBankTM accession number U24275) was cloned from a biopsy taken from a Kaposi's sarcoma skin lesion from an human immunodeficiency virus, type I-infected patient (8Kledal T.N. Rosenkilde M.M. Coulin F. Simmons G. Johnsen A.H. Alouani S. Power C.A. Luttichau H.R. Gerstoft J. Clapham P.R. Clark L.I. Wells T.C. Schwartz T.W. Science. 1997; 277: 1656-1659Crossref PubMed Scopus (427) Google Scholar). Monoiodinated 125I-IL-8,125I-GROα, and myo-[3H]inositol (PT6–271) and Bolton-Hunter reagent for iodination of IP-10 were purchased from Amersham Pharmacia Biotech. AG 1-X8 anion-exchange resin was from Bio-Rad. The Bolton-Hunter reagent was dried by a gentle stream of nitrogen for 30–60 min. 5–10 μg of IP-10 was incubated on ice with 1.5 mCi of Bolton-Hunter reagent in a total volume of 50 μl of 0.1 mm Borat buffer, pH 8.5, for 1 h, and the reaction was terminated by the addition of 0.5 ml of H2O supplemented with 0.1% v/v trifluoroacetic acid. The iodinated chemokines were purified by reverse phase high pressure liquid chromatography. The cDNA encoding the ORF74 receptor was cloned into the eukaryotic expression vector pTEJ-8 (31Johansen T.E. Schøller M.S. Tolstoy S. Schwartz T.W. FEBS Lett. 1990; 267: 289-294Crossref PubMed Scopus (143) Google Scholar). Mutations were constructed by polymerase chain reaction using either the overlap extension method (32Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene ( Amst. ). 1989; 77: 61-68Crossref PubMed Scopus (2641) Google Scholar) for mutations located internal in the receptor or flanking-extended primers for the N-terminal truncated mutation. The polymerase chain reaction products were digested by the appropriate restriction endonucleases, purified, and cloned in the pTEJ8-ORF74. All experiments were performed using thepfu-polymerase, and mutations were verified by restriction endonuclease mapping followed by DNA sequence analysis using the Thermo Sequenase fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP on an Alfexpress DNA sequencer according to the manufacturer's instructions (Amersham Pharmacia Biotech). COS-7 cells were grown at 10% CO2 and 37 °C in Dulbecco's modified Eagle's medium 1885 supplemented with 10% fetal calf serum, 2 mmglutamine, and 0.01 mg/ml gentamicin. Transfection of the COS-7 cells was performed by the calcium phosphate precipitation method (33Rosenkilde M.M. Cahir M. Gether U. Hjorth S.A. Schwartz T.W. J. Biol. Chem. 1994; 269: 28160-28164Abstract Full Text PDF PubMed Google Scholar). COS-7 cells were transferred to culture plates one day after transfection. The number of cells seeded/well was determined by the apparent expression efficiency of the individual clones and was aimed at obtaining 5–10% specific binding of the added radioactive ligand. 3 × 105 and 5 × 105 cells/well were used for mutations with eliminated specific binding. Two days after transfection, cells were assayed by competition binding for 3 h at 4 °C using 12 pm125I-IL-8, 125I-GROα, or125I-IP-10 plus unlabeled ligand in 0.5 ml of 50 mm Hepes buffer, pH 7.4, supplemented with 1 mmCaCl2, 5 mm MgCl2, and 0.5% (w/v) bovine serum albumin. After incubation cells were washed quickly four times in 4 °C binding buffer supplemented with 0.5 mNaCl. Nonspecific binding was determined as the binding in the presence of 0.1 μm unlabeled chemokine. Determinations were made in duplicates. One day after transfection, COS-7 cells (5 × 105 cells/well) were incubated for 24 h with 5 μCi ofmyo-[3H]inositol in 1 ml/well inositol-free Dulbecco's 1885 medium supplemented with 10% fetal calf serum, 2 mm glutamine, and 0.01 mg/ml gentamicin. Cells were washed twice in 20 mm Hepes, pH 7.4, supplemented with 140 mm NaCl, 5 mm KCl, 1 mmMgSO4, 1 mm CaCl2, 10 mm glucose, and 0.05% (w/v) bovine serum albumin and were incubated in 0.8 ml of buffer supplemented with 10 mm LiCl at 37 °C for 90 min in the presence of various concentrations of chemokine. Cells were extracted with 10% ice-cold perchloric acid followed by incubation on ice for 30 min. The resulting supernatant was neutralized with KOH in Hepes buffer, and the generated [3H]inositol phosphates were purified on an AG 1-X8 anion-exchange resin (34Berridge M.J. Dawson M.C. Downes C.P. Heslop J.P. Irvin R.F. Biochem. J. 1983; 212: 473-482Crossref PubMed Scopus (1541) Google Scholar). Determinations were made in duplicates. IC50 and EC50 values were determined by nonlinear regression, andB max values were calculated using the GraphPad-Prism 2 software (GraphPad Software, San Diego). The N-terminal region of the ORF74 receptor is characterized by the occurrence of many acidic residues, whereas multiple basic residues are found at the extracellular ends of TM-V and TM-VI and in extracellular loops 2 and 3 as shown in Fig.1. These two regions were initially targeted for mutagenesis to try to selectively alter the ligand binding without affecting signaling of the virally encoded receptor. Gene dosage experiments showed that deletion of the N-terminal 22 amino acids, including 7 acidic residues (Δ22-N-terminal), did not affect the basal signaling activity of the receptor, i.e. as determined by PI turnover (Fig.2 A). However, neither the agonist GROα nor the inverse agonist IP-10 could affect the high constitutive signaling activity of the mutated receptor (Fig.2 C). A whole panel of human CXC chemokines were tested on the Δ22-N-terminal mutant form of ORF74, but none of them were found to have any influence on its signaling (Fig. 2 F), although several of these chemokines act either as agonists or inverse agonists on the wild type receptor (20Rosenkilde M.M. Kledal T.N. Bräuner-Osborne H. Schwartz T.W. J. Biol. Chem. 1999; 274: 956-961Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). The three radioligands,125I-GROα, 125I-IP-10, and125I-IL-8, which all bind with high affinity to the wild type receptor did not show any binding to the Δ22-N-terminal mutation (Table I).Table ICompetition binding for mutations in the ORF74 receptor125I-GROα125I-IL8125I-IP-10B maxratioIC50(n)B maxIC50(n)B maxIC50(n)B maxGROα/IP-10nmfmol/10 5 cellnmfmol/10 5 cellnmfmol/10 5 cellORF74 wt0.10 ± 0.02(11)44 ± 101.5 ± 0.4(5)42 ± 120.61 ± 0.19(13)28 ± 2.91.57Δ22-N-terminalNB(3)NB(3)NB(3)R208H/R212H0.53 ± 0.19(7)4.5 ± 1.9NB(3)0.73 ± 0.10(9)46 ± 6.20.08R278A/R279A0.10 ± 0.05(4)8.3 ± 0.70.88 ± 0.05(3)2.5 ± 0.23.15 ± 1.21(9)170 ± 650.05L91D0.01 ± 0.003(5)0.28 ± 0.041.3 ± 0.17(3)4.4 ± 1.50.32 ± 0.05(4)3.2 ± 0.520.09N92D0.11 ± 0.07(3)13 ± 4.51.2 ± 0.16(6)8.6 ± 1.50.84 ± 0.15(9)16 ± 60.84S93D0.07 ± 0.03(3)14 ± 62.3 ± 0.93(3)12 ± 1.20.72 ± 0.18(2)11 ± 3.041.33L94D0.02 ± 0.002(5)0.74 ± 0.131.2 ± 0.2(4)3.6 ± 0.680.49 ± 0.25(4)4.9 ± 2.50.15Y128S0.08 ± 0.03(4)5.8 ± 1.31.3 ± 0.39(3)33 ± 110.38 ± 0.14(4)4.3 ± 0.921.36V142D0.08 ± 0.03(3)12 ± 2.11.3 ± 0.17(5)11 ± 2.70.38 ± 0.07(3)7.3 ± 2.71.66N92D/V142D0.05 ± 0.01(3)8.6 ± 2.10.61 ± 0.07(6)2.8 ± 0.670.32 ± 0.04(4)4.2 ± 0.751.73V310N0.09 ± 0.01(2)17 ± 1.92.6 ± 0.28(4)49 ± 50.87 ± 0.19(4)22 ± 8.21.30Homologous competition binding in transiently transfected COS-7 cells with three different radioligands, the agonist 125I-GROα, the inverse agonist 125I-IP10, and the neutral ligand125I-IL8. The table shows IC50 ± S.E. for the indicated number of experiments (n), followed byB max ± S.E. B max ratio indicates the ratio between B max measured with125I-GROα compared to B max measured with125I-IP-10. NB indicates no specific binding. Open table in a new tab Homologous competition binding in transiently transfected COS-7 cells with three different radioligands, the agonist 125I-GROα, the inverse agonist 125I-IP10, and the neutral ligand125I-IL8. The table shows IC50 ± S.E. for the indicated number of experiments (n), followed byB max ± S.E. B max ratio indicates the ratio between B max measured with125I-GROα compared to B max measured with125I-IP-10. NB indicates no specific binding. Based on the observation that IL-8 binding to CXCR2 is highly dependent on the presence of two arginine residues located at the extracellular end of TM-V (35Leong S.R. Kabakoff R.C. Hébert C.A. J. Biol. Chem. 1994; 269: 19343-19348Abstract Full Text PDF PubMed Google Scholar) and that they are among the few residues shared between CXCR2 and ORF74, we substituted these residues as well as two arginine residues located in the corresponding region at the extracellular end of the neighboring TM-VI in ORF74 (Fig.1). The two arginine-substituted ORF74 mutants, like the wild type receptor, displayed high constitutive signaling as demonstrated both by gene-dosing experiments and by the suppressive effect of the inverse agonist, IP-10 (Fig. 2, A, D, and E). However, GROα, which acts as an agonist on the wild type ORF74, did not stimulate signaling detectably above the high basal level in the arginine-substituted ORF74 mutants (Fig. 2, D andE). This is surprising, because GROα did bind with high, normal affinity, albeit with a diminished B maxvalue, to the mutated receptors (4.5 and 8.3 fmol/105 cells for the TM-V mutant (R208H/R212H)-ORF74 and the TM-VI mutant (R278A/R279A)-ORF74, respectively, versus 44 fmol/105 cells for the wild type receptor, Table I). The binding of the neutral ligand IL-8 was totally eliminated in (R208H/R212H)-ORF74, whereas in (R278A/R279A)-ORF74, IL-8 binding was not affected in respect of affinity (K d = 0.88 nm versus 1.5 nm for wild type receptor) but was severely decreased in respect ofB max (2.5 fmol/105 cells as compared with 42 fmol/105 cells for the wild type ORF74) (Table 1). In contrast, for the inverse agonist, IP-10-increasedB max values were observed in both of the arginine-substituted ORF74 mutants (46 and 170 fmol/105cells for (R208H/R212H]-ORF74 and [R278A/R279A)-ORF74, respectively,versus 28 fmol/105 cells for the wild type receptor) with preserved high affinity IP-10 binding (Table I). A number of residues in the transmembrane segments have been implicated as being important for the signal transduction process in 7TM receptors in general. As shown in Fig. 1, many of these residues have been mutated by the virus in ORF74. In an attempt to localize the structural basis for the high constitutive activity of the virally encoded receptor, we re-introduced "normal" 7TM residues at a number of places in the transmembrane segments of ORF74. Most of the substitutions in the transmembrane segments of ORF74 did not affect the receptor neither in respect of ligand binding nor in respect of constitutive signaling. Thus, in gene dosage experiments the (Y128S)-, (V142D)-, (N92D/V142D)-, and (V310N)-ORF74 mutants all had similar basal activity as compared with wild type ORF74 (Fig.3 A). These mutants were also affected by chemokine ligands in a similar manner as the wild type receptor was as they were unaffected by IL-8, stimulated by GROα, and inhibited by IP-10 (Fig. 3 B). One of the mutants, (Y128S)-ORF74, did show a slightly higher constitutive activity and was affected less by both GROα and IP-10 than the wild type receptor. The competition binding analysis for these mutations also gave results rather similar to those observed in the wild type receptor, although the expression level of (Y128S)-, (V142D)-, and (N92D/V142D)-ORF74 was lower for all tested radioactive ligands (TableI). Further mutational analysis was performed in TM-II. This segment normally holds AspII:10, which is highly important for signaling in 7TMs in general. Asn92 in ORF74 was the most obviously candidate for a mutated AspII:10. However, "reintroduction" of an Asp at this position either alone or in combination with an Asp in position III:25 in the "DRY" motif did not have a major effect on either ligand binding (Table I) or receptor signaling (Fig. 3 and4). We then decided to try to re-introduce an Asp residue at other positions in TM-II around Asn92. Substitution of the other polar residue in this region, Ser93, with an Asp had as little effect on receptor signaling and ligand binding as the substitution of Asn92(Fig. 4 A). Surprisingly, introduction of an Asp residue for either Leu91 or Leu94, both of which would be expected to be facing more or less into the lipid bilayer (Fig. 1), eliminated the high constitutive activity of the ORF74 receptor while preserving its ability to be stimulated by GROα (Fig. 4). Because of the low basal activity, the relative stimulatory effect of GROα was even larger, ∼3-fold, in the (L91D)- and (L94D)-ORF74 constructs as compared with the 2-fold stimulation found in the wild type receptor. Also because of the low basal activity, which was similar to that observed in cells transfected with the empty expression vector, IP-10 was unable to show any inverse agonistic activity in these two mutants (Fig. 4 D). The expression level for the four different Asp mutations in TM-II was relatively similar, between 3.6 and 12 fmol/105 cells, but less than the expression level of the wild type receptor as determined by the B max values using the neutral ligand125I-IL-8 (Table I). Similarly, theB max values for 125I-IP-10 were comparable in this group of mutants, between 3.2 and 16 fmol/105 cells (Table I). In contrast, theB max values for 125I-GROα were very low, 0.28 and 0.74 fmol/105 cells, for the two mutants in which the constitutive activity had been silenced, (L91D)- and (L94D)-ORF74 as compared with the B max for GROα in the (N92D)- and (S93D)-ORF74 and the wild type ORF74, 13, 14, and 44 fmol/105 cells (Table I). Nevertheless, despite the apparent low B max values, GROα was able to efficiently stimulate signaling of the (L91D)- and (L94D)-ORF74 constructs. In the present study three distinct phenotypes of ORF74 are generated through targeted mutagenesis directed toward either presumed ligand binding epitopes in the extracellular domains or toward the seven helical bundle, which is responsible for the signal transduction: 1) a receptor with eliminated ligand binding but with maintained high constitutive activity; 2) a receptor with selective elimination of agonist action, GROα, but with preserved inverse agonist action, IP-10, and also with preserved high constitutive activity; and 3) a receptor with eliminated constitutive activity but with preserved ligand binding and importantly with maintained ability to be stimulated by agonists. The elimination of all chemokine binding through truncation of the N terminus of the receptor is in agreement with the observation that peptide and protein ligands of the 7TM receptors in general often have important interactions with the extracellular segments (36Schwartz T.W. Curr. Opin. Biotechnol. 1994; 5: 434-444Crossref PubMed Scopus (280) Google Scholar). Especially in chemokine receptors, ligand-receptor interactions have often been located to their N-terminal segment (37Monteclaro F.S. Charo I.F. J. Biol. Chem. 1996; 271: 19084-19092Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 38Farzan M. Choe H. Martin C.A. Sun Y. Gerard N. Sodroski J. Gerard C. J. Biol. Chem. 1997; 272: 6854-6857Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 39Blanpain C. Doranz B.J. Vakili J. Rucker J. Govaerts C. Baik S.S. Lorthioir O. Migeotte I. Libert F. Baleux F. Vassart G. Doms R.W. Parmentier M. J. Biol. Chem. 1999; 274: 34719-34727Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Most significantly, Handel and co-workers (40Hemmerich S. Paavola C. Bloom A. Bhakta S. Freedman R. Grunberger D. Krstenansky J. Lee S. McCarley D. Mulkins M. Wong B. Pease J. Mizoue L. Mirzadegan T. Polsky I. Thompson K. Handel T.M. Jarnagin K. Biochemistry. 1999; 38: 13013-13025Crossref PubMed Scopus (141) Google Scholar) have recently by NMR demonstrated the precise molecular binding mode of a peptide from the N-terminal segment of the CCR2 receptor to a structurally complementary groove on the main agonist for this receptor, MCP-1 (monocyte chemotactic protein-1). In the case of ORF74, Gershengorn and co-workers (41Ho H.H. Du D. Gershengorn M.C. J. Biol. Chem. 1999; 274: 31327-31332Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) recently found in analogy with the observation in the present study that truncation of the N terminus eliminated agonist binding. Importantly, in both studies it was found that the high constitutive activity of the N-terminally truncated ORF74 receptor was similar to that of the wild type receptor (Fig. 2). In an attempt to affect the binding and action of various types of chemokine ligands selectively, we focused on the extracellular ends of TM-V and -VI, because these epitopes are known often to be involved in both agonist and antagonist binding in 7TM receptors (36Schwartz T.W. Curr. Opin. Biotechnol. 1994; 5: 434-444Crossref PubMed Scopus (280) Google Scholar). Specifically in CXCR1 and -2, which are the endogenous receptors with ligand binding profiles most similar to that of ORF74, two Arg residues are located at positions V:01 and V:05 facing the main ligand binding crevice and are known to be crucially involved in the binding of IL-8 (35Leong S.R. Kabakoff R.C. Hébert C.A. J. Biol. Chem. 1994; 269: 19343-19348Abstract Full Text PDF PubMed Google Scholar). These residues are conserved in ORF74, and we find here that substitution of the two Args with His residues in ORF74 also eliminated binding of IL-8, which is a neutral ligand in this virally encoded receptor. However, from a functional point of view this double mutation was even more interesting than expected, because it surprisingly eliminated the action of the agonist chemokine GROα without affecting the binding or action of the inverse agonist chemokine, IP-10 (Fig. 2). GROα still bound to the mutated receptor, albeit with a 5-fold reduced affinity and a 10-fold reduced B max. The inability of the R208H/R212H mutation to be stimulated by GROα cannot be explained simply by the low B max for the agonist, because, for example the L91D and L94D mutants display even lowerB max values for GROα both in total numbers and relative to the B max values for the inverse agonist IP-10 (Table I). Instead, an explanation could be that the double mutation eliminates a ligand-receptor interaction, which although being essential for the binding of the neutral ligand, IL-8, is only of limited importance for the binding of the agonist GROα but is an interaction that is essential for the function of GROα as an agonist. Importantly, the mutated receptor signals with high constitutive activity and this activity can be blocked normally by the inverse agonist peptide IP-10. Thus this mutation provides a unique tool to determine the relative importance of agonist versusinverse agonist regulation of ORF74 activity for the angiogenic property of the virally encoded receptor to be determined, for example in transgenic animals (1Yang Y.T. Chen S.C. Leach M.W. Manfra D. Homey B. Wiekowski M. Sullivan L. Jenh C.H. Narula S.K. Chensue S.W. Lira S.A. J. Exp. Med. 2000; 191: 445-454Crossref PubMed Scopus (358) Google Scholar). The virally encoded receptor differs structurally at many positions from the classical 7TM pattern of conserved residues. Based on these differences, a number of substitutions were made in the transmembrane helical bundle to normalize the receptor structurally and thereby try to normalize the high constitutive activity of ORF74. For example, at the intracellular end of TM-III in most 7TM receptors is found the tri-peptide sequence Asp-Arg-Tyr (DRY) of which the Asp and Arg are highly conserved and crucially involved in receptor G-protein interaction and signaling (26Scheer A. Fanelli F. Costa T. Cotecchia S. EMBO J. 1996; 15: 3566-3578Crossref PubMed Scopus (361) Google Scholar, 27Scheer A. Fanelli F. Costa T. Benedetti P. Cotecchia S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 808-813Crossref PubMed Scopus (200) Google Scholar). In ORF74 the Asp in the DRY sequence is substituted with a Val. In the middle of TM-III of ORF74 a large aromatic side chain is located at position III:11 (Tyr128), which in the angiotensin AT1 receptor (28Balmforth A.J. Lee A.J. Warburton P. Donelly D. Ball S.G. J. Biol. Chem. 1997; 272: 4245-4251Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) and in the bradykinin receptor (29Marie J. Kock C. Pruneau D. Paquet J.L. Groblewski T. Larguier R. Lombard C. Deslauriers B. Maigret B. Bonnafous J. Mol. Pharmacol. 1999; 55: 92-101Crossref PubMed Scopus (58) Google Scholar) creates a receptor phenotype dominated by high constitutive activity. In TM-VII the highly conserved Asn at position VII:16, which normally is believed to be involved in creating an important interhelical hydrogen bond network, is in ORF74 substituted with a Val lacking the ability to form hydrogen bonds. In TM-II the functionally highly important Asp residue is substituted with either an Asn or a Ser residue depending on how the sequence alignment is performed. However, normalization of ORF74 at these positions did not affect the high constitutive signaling of the receptor (Fig. 4). This was most surprising in the case of the DRY sequence, because substitution of the Asp in several cases, one of which is CXCR2, has created highly constitutively active receptors (26Scheer A. Fanelli F. Costa T. Cotecchia S. EMBO J. 1996; 15: 3566-3578Crossref PubMed Scopus (361) Google Scholar, 27Scheer A. Fanelli F. Costa T. Benedetti P. Cotecchia S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 808-813Crossref PubMed Scopus (200) Google Scholar, 42Burger M. Burger J.A. Hoch R.C. Oades Z. Takamori H. Schraufstatter I.U. J. Immunol. 1999; 163: 2017-2022PubMed Google Scholar). In the CXCR2 study, it was even argued that this substitution probably was the reason for the high constitutive activity of ORF74 (42Burger M. Burger J.A. Hoch R.C. Oades Z. Takamori H. Schraufstatter I.U. J. Immunol. 1999; 163: 2017-2022PubMed Google Scholar); however, the mutation performed here in the ORF74 could not confirm that notion. Thus, the rational approach to normalizing ORF74 signaling was not successful in our hands. Nevertheless, introduction of an Asp residue at either position 91 or position 94 in TM-II surprisingly created the desired phenotype,i.e. low constitutive signaling activity combined with the ability to be stimulated by agonists. Because of the poorly conserved primary structure around TM-II, it was unclear how the sequence of ORF74 was best aligned with the consensus 7TM sequences, which basically only dictates the presence of an Asp residue located at position II:10 in an otherwise rather hydrophobic segment. Most likely it is either the Asn92 or perhaps the Ser93residue that corresponds to the TM-II Asp found in almost all 7TM receptors. However, because the introduction of an Asp at these positions had almost no effect on the basal signaling of ORF74, the neighboring positions were also probed and with a surprisingly positive result (Fig. 1). The L91D and L94D substitutions abolished the constitutive activity, without influencing the surface expression of the receptor more than introduction of Asps at the neighboring positions 92 and 93 did; the latter two mutations were displayed as high basal activity as observed in the wild type receptor. From the helical wheel diagram of TM-II, it is predicted that Leu91and Leu94 are both located on a hydrophobic, presumed membrane-exposed face of this helix (Fig. 1). It is unclear what the structural basis for the effect of these substitutions is. It could be envisioned that introduction of the polar, potentially charged Asp residue in the middle of the hydrophobic face would destabilize the receptor structure. However, how this could lead to diminished basal signaling but preserved responsiveness to the agonist is unclear. It has been suggested that ORF74 through its ligand-independent high constitutive activity should be causatively involved in the development of Kaposi's sarcoma as well as certain types of B-cell lymphomas (19Arvanitakis L. Geras-Raaka E. Varma A. Gershengorn M.C. Cesarman E. Nature. 1997; 385: 347-350Crossref PubMed Scopus (575) Google Scholar, 21Bais C. Santomasso B. Coso O. Arvanitakis L. Raaka E.G. Gutkind J.S. Asch A.S. Cesarman E. Gerhengorn M.C. Mesri E.A. Nature. 1998; 391: 86-89Crossref PubMed Scopus (749) Google Scholar). Recently it was elegantly shown by Lira and co-workers (1Yang Y.T. Chen S.C. Leach M.W. Manfra D. Homey B. Wiekowski M. Sullivan L. Jenh C.H. Narula S.K. Chensue S.W. Lira S.A. J. Exp. Med. 2000; 191: 445-454Crossref PubMed Scopus (358) Google Scholar) that transgenic mice expressing the wild type ORF74 receptor under control of the CD2 promoter develop Kaposi's sarcoma-like lesions. This obviously strongly supports a pathogenic connection between ORF74 from HHV-8 and Kaposi's sarcoma. However, it is still unclear whether it is merely the constitutive activity of the virus-encoded oncogene as such that causes the lesions or whether the carefully evolved control of this activity by angiogenic as well as angiostatic or modulator chemokines is involved in the development of these specially highly vascularized tumors. It should also be noted that Kaposi's sarcoma predominantly develops in immune compromised individuals and that the function of ORF74 in the life circle of HHV-8 may be more subtle in the normal HHV-8-infected individuals. Nevertheless, expression of the various ORF74 mutants with and without high constitutive activity as well as with and without chemokine or selective angiogenic chemokine regulation in transgenic animals should be able to clarify the molecular mechanism exploited by HHV-8 in cell transformation and angiogenesis. We thank Lisbet Elbak for excellent technical assistance.

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