Previously it was shown that the HHV-8-encoded chemokine receptor ORF74 shows considerable agonist-independent, constitutive activity giving rise to oncogenic transformation (Arvanitakis, L., Geras-Raaka, E., Varma, A., Gershengorn, M. C., and Cesarman, E. (1997) Nature 385, 347-350). In this study we report that a second viral-encoded chemokine receptor, the human cytomegalovirus-encoded US28, also efficiently signals in an agonist-independent manner. Transient expression of US28 in COS-7 cells leads to the constitutive activation of phospholipase C and NF-B signaling via G q/11 proteindependent pathways. Whereas phospholipase C activation is mediated via G␣ q/11 subunits, the activation of NF-B strongly depends on ␤␥ subunits with a preference for the ␤ 2 ␥ 1 dimer. The CC chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and MCP-1 (monocyte chemotactic protein-1) act as neutral antagonists at US28, whereas the CX 3 C chemokine fractalkine acts as a partial inverse agonist with IC 50 values of 1-5 nM. Our data suggest that a high level of constitutive activity might be a more general characteristic of viral G protein-coupled receptors and that human cytomegalovirus might exploit this G protein-coupled receptor property to modulate the homeostasis of infected cells via the early gene product US28.

Human cytomegalovirus (HCMV) is a widely spread herpesvirus, suggested to play a role in tumor progression. US28, a chemokine receptor encoded by HCMV, binds a broad spectrum of chemokines and constitutively activates various pathways linked to proliferation. Our studies reveal that expression of US28 induces a proangiogenic and transformed phenotype by up-regulating the expression of vascular endothelial growth factor and enhancing cell growth and cell cycle progression. US28-expressing cells promote tumorigenesis when injected into nude mice. The G proteinuncoupled constitutively inactive mutant of US28, induces delayed and attenuated tumor formation, indicating the importance of constitutive receptor activity in the early onset of tumor development. Importantly, also in glioblastoma cells infected with the newly isolated clinical HCMV strain Titan, US28 was shown to be involved in the HCMV-induced angiogenic phenotype. Hence, the constitutively activated chemokine receptor US28 might act as a viral oncogene and enhance and͞or promote HCMV-associated tumor progression.cancer ͉ G protein-coupled receptor ͉ VEGF ͉ viral infection ͉ drug target

Sphingolipids are a class of biologically active lipids that have a role in multiple biological processes including inflammation. Sphingolipids exert their functions by direct signaling or through signaling by their specific receptors. Phosphorylated FTY720 (FTY720P) is a sphingosine 1-phosphate (S1P) analogue that is currently in trial for treatment of multiple sclerosis (MS), which targets all S1P receptors but S1P(2). To date, however, it remains unknown whether FTY720P may exert direct anti-inflammatory effects within the central nervous system (CNS), because data concerning S1P receptor expression and regulation under pathological conditions in the human brain are lacking. To investigate potential regulation of S1P receptors in the human brain during MS, we performed immunohistochemical analysis of S1P receptor 1 and 3 expression in well-characterized MS lesions. A strong increase in S1P receptor 1 and 3 expression on reactive astrocytes was detected in active and chronic inactive MS lesions. In addition, we treated primary cultures of human astrocytes with the proinflammatory cytokine tumor necrosis factor-alpha to identify the regulation of S1P(1/3) on astrocytes under pathological conditions. Importantly, we demonstrate that FTY720P exerts an anti-inflammatory action on human astrocytes by limiting secretion of proinflammatory cytokines. Our data demonstrate that reactive astrocytes in MS lesions and cultured under proinflammatory conditions strongly enhance expression of S1P receptors 1 and 3. Results from this study indicate that astrocytes may act as a yet-unknown target within the CNS for the anti-inflammatory effects observed after FTY720P administration in the treatment of MS.

Epstein-Barr virus (EBV) infection is associated with many lymphoproliferative diseases, such as infectious mononucleosis andBurkitt's lymphoma. Consequently, EBV is one of the most extensively studied herpesviruses. Surprisingly, a putative G protein-coupled receptor (GPCR) gene of EBV, BILF1, has hitherto escaped attention, yet BILF1-like genes are conserved among all known lymphocryptovirus species, suggesting that they play a pivotal role in viral infection. To determine the function of EBV BILF1, the activity of this gene and its products was studied. BILF1-specific mRNA was detected in various EBV-positive cell types and found to be expressed predominantly during the immediate early and early phases of infection in vitro. Interestingly, in COS-7 cells transfected with BILF1 expression constructs, a decrease in forskolin-induced CRE-mediated transcription was measured, as well as an increase in NF-B-mediated transcription. In contrast, CREmediated transcription was increased in EBV-positive Burkitt's lymphoma cells as well as EBV-positive lymphoblastoid B cells transfected with BILF1, whereas NF-B-mediated transcription levels remained unaffected in these cells. All observed activities were sensitive to treatment with pertussis toxin, indicating that the BILF1-encoded protein mediates these activities by coupling to G proteins of the G i/o class. Finally, reduced levels of phosphorylated RNA-dependent antiviral protein kinase were observed in COS-7 and Burkitt's lymphoma cells transfected with BILF1. Neither of the observed effects required a ligand to interact with the BILF1 gene product, suggesting that BILF1 encodes a constitutively active GPCR capable of modulating various intracellular signaling pathways.

Allosteric modulators for the Gi-coupled A3 adenosine receptor (AR) are of considerable interest as therapeutic agents and as pharmacological tools to probe various signaling pathways. In this study, we initially characterized the effects of several imidazoquinolinamine allosteric modulators (LUF5999, LUF6000 and LUF6001) on the human A3 AR stably expressed in CHO cells using a cyclic AMP functional assay. These modulators were found to affect efficacy and potency of the agonist Cl-IB-MECA differently. LUF5999 (2-cyclobutyl derivative) enhanced efficacy but decreased potency. LUF6000 (2-cyclohexyl derivative) enhanced efficacy without affecting potency. LUF6001 (2-H derivative) decreased both efficacy and potency. We further compared the agonist enhancing effects of LUF6000 in several other A3 AR-mediated events. It was shown that although LUF6000 behaved somewhat differently in various signaling pathways, it was more effective in enhancing the effects of low-efficacy than of high-efficacy agonists. In an assay of cyclic AMP accumulation, LUF6000 enhanced the efficacy of all agonists examined, but in the membrane hyperpolarization assay, it only enhanced the efficacy of partial agonists. In calcium mobilization, LUF6000 did not affect the efficacy of the full agonist NECA but was able to switch the nucleoside antagonist MRS542 into a partial agonist. In translocation of β-arrestin2, the agonist-enhancing effect LUF6000 was not pronounced. In an assay of ERK1/2 phosphorylation LUF6000 did not show any effect on the efficacy of Cl-IB-MECA. The differential effects of LUF6000 on the efficacy and potency of the agonist Cl-IB-MECA in various signaling pathway were interpreted quantitatively using a mathematical model.

BACKGROUND AND PURPOSEThe chemokine receptor CXCR3 is a GPCR found predominantly on activated T cells. CXCR3 is activated by three endogenous peptides; CXCL9, CXCL10 and CXCL11. Recently, a small-molecule agonist, VUF10661, has been reported in the literature and synthesized in our laboratory. The aim of the present study was to provide a detailed pharmacological characterization of VUF10661 by comparing its effects with those of CXCL11. EXPERIMENTAL APPROACHAgonistic properties of VUF10661 were assessed in a chemotaxis assay with murine L1.2 cells transiently transfected with cDNA encoding the human CXCR3 receptor and in binding studies, with [ KEY RESULTSVUF10661 acted as a partial agonist in CXCR3-mediated chemotaxis, bound to CXCR3 in an allosteric fashion in ligand binding assays and activated Gi proteins with the same efficacy as CXCL11 in the [ CONCLUSIONS AND IMPLICATIONSVUF10661, like CXCL11, activates both G protein-dependent and -independent signalling via the CXCR3 receptor, but probably exerts its effects from an allosteric binding site that is different from that for CXCL11. It could stabilize different receptor and/or b-arrestin conformations leading to differences in functional output. Such ligand-biased signalling might offer interesting options for the therapeutic use of CXCR3 agonists. LINKED ARTICLEThis article is commented on by O'Boyle, pp. 895-897 of this issue. To view this commentary visit http://dx

The G protein-coupled receptor encoded by Kaposi's sarcoma-associated herpesvirus, also referred to as ORF74, has been shown to stimulate oncogenic and angiogenic signaling pathways in a constitutively active manner. The biochemical routes linking ORF74 to these signaling pathways are poorly defined. In this study, we show that ORF74 constitutively activates p44/p42 mitogen-activated protein kinase (MAPK) and Akt via G iand phospholipase C (PLC)-mediated signaling pathways. Activation of Akt by ORF74 appears to be phosphatidylinositol 3-kinase (PI3-K) dependent but, interestingly, is also mediated by activation of protein kinase C (PKC) and p44/p42 MAPK. ORF74 may signal to Akt via p44/p42 MAPK, which can be activated by G i , through activation of PI3-K or through PKC via the PLC pathway. Signaling of ORF74 to these proliferative and antiapoptotic signaling pathways can be further modulated positively by growth-related oncogene (GRO␣/ CXCL1) and negatively by human gamma interferon-inducible protein 10 (IP-10/CXCL10), thus acting as an agonist and an inverse agonist, respectively. Despite the ability of the cytomegalovirus-encoded chemokine receptor US28 to constitutively activate PLC, this receptor does not increase phosphorylation of p44/p42 MAPK or Akt in COS-7 cells. Hence, ORF74 appears to signal through a larger diversity of G proteins than US28, allowing it to couple to proliferative and antiapoptotic signaling pathways. ORF74 can therefore be envisioned as an attractive target for novel treatment of Kaposi's sarcoma.

The human cytomegalovirus (HCMV) UL33 gene is conserved among all ␤-herpesviruses and encodes a protein that shows sequence similarity with chemokine receptors belonging to the family of G protein-coupled receptors. Here, we show that HCMV UL33 is predominantly transcribed as a spliced mRNA of which the 5 terminus is localized 55 bp upstream of the start codon. Like its homolog from rat cytomegalovirus (RCMV), R33, UL33 activates multiple signaling pathways in a ligandindependent manner. Although both receptors constitutively activate phospholipase C via G q/11 , and partially via G i/o -mediated pathways, they exhibit profound differences in the modulation of cAMP-responsive element (CRE) activation. R33 constitutively inhibits, whereas UL33 constitutively enhances CRE-mediated transcription. For R33, the inhibition of CRE-driven transcription is entirely G i/o -mediated. For UL33, however, CREmediated transcription is modulated not only through coupling to G␣ i/o but also through coupling to G␣ s. In addition, UL33 was found to enhance CRE activation through the Rho/p38 pathway, via G␤␥. Interestingly, by studying chimeric UL33/R33 proteins, we found the Cterminal cytoplasmic tail of UL33, but not that of R33, to be responsible for the activation of G i/o proteins. A UL33-deficient variant of HCMV was generated to analyze UL33-signaling properties in a physiologically relevant model system. Data obtained with infected cells show that HCMV induces CRE activation, and this effect is, at least in part, dependent on UL33 expression. Taken together, our data indicate that constitutive signaling of UL33 differs from that of R33 by promiscuous activation of G proteins of the G q , G i/o , as well as G s class. Thus, HCMV may effectively use UL33 to orchestrate multiple signaling networks within infected cells.