Constitutive Signaling by Kaposi's Sarcoma-associated Herpesvirus G-protein-coupled Receptor Desensitizes Calcium Mobilization by Other Receptors

Abstract

We coexpressed Kaposi's sarcoma-associated herpesvirus G protein-coupled receptors (KSHV-GPCRs) with thyrotropin-releasing hormone (TRH) receptors or m1-muscarinic-cholinergic receptors in Xenopus oocytes and in mammalian cells. In oocytes, KSHV-GPCR expression resulted in pronounced (81%) inhibition (heterologous desensitization) of Ca 2؉ -activated chloride current responses to TRH and acetylcholine. 45 Ca 2؉ efflux. In KSHV-GPCR-expressing oocytes, responses to microinjected inositol 1,4,5-trisphosphate were inhibited by 74%, and this effect was partially reversed by interferon-␥-inducible protein 10. Treatment with thapsigargin suggested that the pool of calcium available for mobilization by TRH was decreased in oocytes coexpressing KSHV-GPCRs. These results suggest that constitutive signaling by KSHV-GPCR causes heterologous desensitization of responses mediated by other receptors, which signal via the phosphoinositide/calcium pathway, which is caused by depletion of intracellular calcium pools. KSHV-GPCR,1 the gene product of ORF74 in the KSHV (human herpesvirus 8) genome, is a GPCR homologous to human chemokine CXC receptor 2 (1, 2). Our group demonstrated that rodent fibroblasts expressing KSHV-GPCRs formed tumors in mice (3). Recently, transgenic mice in which the KSHV-GPCR gene was regulated by the human CD2 enhancer/promoter and locus control region were shown to develop angioproliferative lesions that resembled the lesions of KS (4). These reports make the mechanism of action of KSHV-GPCR interesting in terms of tumorigenesis. We reported that KSHV-GPCR exhibited marked constitutive, agonist-independent activity in mammalian cells (2). We also demonstrated that the constitutive activity of the viral GPCR could be inhibited by IP-10, acting as an inverse agonist (5). These results were later confirmed by Rosenkilde et al. (6). Last, we showed that signaling by KSHV-GPCR could be inhibited by GPCR-specific protein kinases and suggested that this may represent a form of homologous desensitization of this viral receptor (7). However, the effect of the constitutive activity of KSHV-GPCR on signaling by other receptors (heterologous desensitization) has not been previously investigated.To investigate the effect of constitutive signaling by KSHV-GPCR on other receptors that utilize the phosphoinositide/ calcium signaling pathway, we studied the behavior of this receptor in Xenopus oocytes and in mammalian cells. Our results show that KSHV-GPCR causes marked desensitization of responses mediated by TRH-Rs and m1-Rs by inhibiting InsP 3 -stimulated mobilization of Ca 2ϩ . EXPERIMENTAL PROCEDURESXenopus laevis Oocytes-Defolliculated oocytes were obtained from mature Xenopus females, essentially as previously described (8). In vitro transcribed cRNAs for wild type TRH-Rs, m1-Rs (1 ng/oocyte), gastrin-releasing peptide (GRP) receptors (0.23 ng/oocyte), and KSHVGPCRs (1-3 ng/oocyte) were injected 48 h before the assay as described previously (9 -11).Electrophysiology-Two-electrode voltage clamp measurements wer...

Carboxyl Tail Cysteine Mutants of the Thyrotropin-Releasing Hormone Receptor Type 1 Exhibit Constitutive Signaling: Role of Palmitoylation

“…We described previously heterologous desensitization caused by a constitutively active 7TMR, such as a viral 7TMR (Lupu-Meiri et al, 2001), as a sensitive reporter of constitutive signaling in oocytes. Coexpression of C335G/C337G/K338Stop and the muscarinic M1 receptor caused more than 40% desensitization of M1 receptors (Table 2).…”

Piracy of Prostaglandin E2/EP Receptor–Mediated Signaling by Kaposi's Sarcoma-Associated Herpes Virus (HHV-8) for Latency Gene Expression: Strategy of a Successful Pathogen

“…Previous reports have indicated the role of Ca 2+ in KSHV lytic cycle (17-20). In contrast, our studies showing the down-regulation of LANA-1 expression by EP1 receptor antagonist, the blockage of supernatant induced [Ca 2+ ] i signal by EP1 antagonist, and the down-regulation PGE2 induced LANA-1 promoter activity by calcium chelators are the first demonstration of a role for [Ca 2+ ] i in KSHV latency program.…”

Lysophosphatidic Acid Decreases the Nuclear Localization and Cellular Abundance of the p53 Tumor Suppressor in A549 Lung Carcinoma Cells

“…First, it is quite possible that overexpressed LPA receptors signal in a ligand-independent manner. There are numerous examples of G protein -coupled receptors that are constitutively active, especially those encoded by human viruses, such as Kaposi's sarcoma virus, EBV, and cytomegalovirus (61)(62)(63). Indeed, overexpression of LPA 1 in Schwann cells protects them from apoptosis induced by serum deprivation, with addition of LPA having little additional effect (64).…”