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. 1998 Jul 20;188(2):405-8.
doi: 10.1084/jem.188.2.405.

Human interferon-gamma-inducible protein 10 (IP-10) inhibits constitutive signaling of Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor

E Geras-Raaka  1 , A Varma, H Ho, I Clark-Lewis, M C Gershengorn
Affiliations

Human interferon-gamma-inducible protein 10 (IP-10) inhibits constitutive signaling of Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor

E Geras-Raaka et al. J Exp Med. .

Abstract

A G protein-coupled receptor (GPCR) is encoded within the genome of Kaposi's sarcoma- associated herpesvirus (KSHV)/human herpesvirus 8, a virus that may be involved in the pathogenesis of Kaposi's sarcoma and primary effusion lymphomas. KSHV-GPCR exhibits constitutive signaling activity that causes oncogenic transformation. We report that human interferon (IFN)-gamma-inducible protein 10 (HuIP-10), a C-X-C chemokine, specifically inhibits signaling of KSHV-GPCR. In contrast, monokine induced by IFN-gamma (HuMig), which like HuIP-10 is an agonist of C-X-C chemokine receptor 3, does not inhibit KSHV-GPCR signaling. Moreover, HuIP-10, but not HuMig, inhibits KSHV-GPCR-induced proliferation of NIH 3T3 cells. These results show that HuIP-10 is an inverse agonist that converts KSHV-GPCR from an active to an inactive state. Thus, a human chemokine inhibits constitutive signaling and cellular proliferation that is mediated by a receptor encoded by a human disease-associated herpesvirus.

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Figures

Figure 1
Figure 1
Effects of HuIP-10 (filled squares), HuMig (filled circles), MuIP-10 (open squares), and MuMig (open circles) on constitutive KSHV-GPCR signaling. Inositol phosphate accumulation in COS-1 cells transiently transfected with plasmid encoding KSHV-GPCR was measured as described in Materials and Methods. Chemokine analogues were added at the concentrations indicated 15 min before LiCl (10 mM). Untransfected cells and cells transfected with plasmid without DNA encoding KSHV-GPCR (“mock”) or with plasmid encoding the receptor for thyrotropin-releasing hormone were studied in parallel. There was no effect of any of these chemokines on inositol phosphate production in untransfected cells, in mock-transfected cells, or in cells expressing thyrotropin-releasing hormone receptors (not shown). The dose of HuIP-10 that caused 50% inhibition of inositol phosphate formation was 15 nM (7.2–32 nM, 95% confidence interval). The data represent the mean ± SD of triplicate determinations in a representative of three experiments.
Figure 2
Figure 2
Effects of truncations of the NH2 termini of HuIP-10 and KSHV-GPCR on inhibition of KSHV-GPCR signaling. Inositol phosphate accumulation in COS-1 cells transiently transfected with plasmid encoding KSHV-GPCR or KSHV-GPCR(Δ2–11) was measured as described in Materials and Methods. HuIP-10, HuIP-10(4–77), or HuIP-10(9–77) was added at the concentrations indicated 15 min before LiCl (10 mM). (A) Effect of truncating the NH2 terminus of the ligand HuIP-10. The data represent the mean ± SD of triplicate determinations in a representative of three experiments. Filled squares, HuIP-10; open circles, HuIP-10(4–77); filled triangles, HuIP-10(9–77). (B) Effect of truncating the NH2 terminus of KSHV-GPCR. The data represent the mean ± SD of triplicate determinations in a representative experiment in which two plasmid clones encoding KSHV-GPCR(Δ2–11) were tested. WT, wild-type.
Figure 2
Figure 2
Effects of truncations of the NH2 termini of HuIP-10 and KSHV-GPCR on inhibition of KSHV-GPCR signaling. Inositol phosphate accumulation in COS-1 cells transiently transfected with plasmid encoding KSHV-GPCR or KSHV-GPCR(Δ2–11) was measured as described in Materials and Methods. HuIP-10, HuIP-10(4–77), or HuIP-10(9–77) was added at the concentrations indicated 15 min before LiCl (10 mM). (A) Effect of truncating the NH2 terminus of the ligand HuIP-10. The data represent the mean ± SD of triplicate determinations in a representative of three experiments. Filled squares, HuIP-10; open circles, HuIP-10(4–77); filled triangles, HuIP-10(9–77). (B) Effect of truncating the NH2 terminus of KSHV-GPCR. The data represent the mean ± SD of triplicate determinations in a representative experiment in which two plasmid clones encoding KSHV-GPCR(Δ2–11) were tested. WT, wild-type.
Figure 3
Figure 3
Inhibition of constitutive KSHV-GPCR signaling and DNA synthesis in NIH 3T3 mouse fibroblasts. (A) Inositol phosphate accumulation in NIH 3T3 cells stably expressing KSHV-GPCRs (reference 7). HuIP-10, HuMig, MuIP-10, or MuMig (300 μM) was added 15 min before the addition of LiCl (10 mM). The data represent the mean ± SD of triplicate determinations in one of two experiments. (B) Dose-dependent effects of HuIP-10 on inositol phosphate formation (open circles) and DNA synthesis (filled squares). The concentration of HuIP-10 required for 50% inhibition (IC50) of inositol phosphate second messenger formation is 39 nM (9–160 nM; 95% confidence interval), and for 50% inhibition of DNA synthesis is 29 nM (8–110 nM, 95% confidence interval). The data represent the mean ± SE of triplicate determinations in three experiments.
Figure 3
Figure 3
Inhibition of constitutive KSHV-GPCR signaling and DNA synthesis in NIH 3T3 mouse fibroblasts. (A) Inositol phosphate accumulation in NIH 3T3 cells stably expressing KSHV-GPCRs (reference 7). HuIP-10, HuMig, MuIP-10, or MuMig (300 μM) was added 15 min before the addition of LiCl (10 mM). The data represent the mean ± SD of triplicate determinations in one of two experiments. (B) Dose-dependent effects of HuIP-10 on inositol phosphate formation (open circles) and DNA synthesis (filled squares). The concentration of HuIP-10 required for 50% inhibition (IC50) of inositol phosphate second messenger formation is 39 nM (9–160 nM; 95% confidence interval), and for 50% inhibition of DNA synthesis is 29 nM (8–110 nM, 95% confidence interval). The data represent the mean ± SE of triplicate determinations in three experiments.
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