Kaposi's Sarcomaassociated Herpesvirus GPCR Elizabeth Geras-Raaka and Marvin C. Gershengorn* Department of Medicine, Division of Molecular Medicine, Weill Medical College and Graduate School of Medical Sciences of Cornell University, 1300 York Avenue, New York, NY 10021, USA * corresponding author tel: 212-746-6275, fax: 212-746-6289, e-mail:
[email protected] DOI: 10.1006/rwcy.2000.22013.
SUMMARY
BACKGROUND
Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus 8), which is a virus that appears to be etiologic for Kaposi's sarcoma, primary effusion lymphomas, and multicentric Castleman's disease in humans, encodes a G protein-coupled receptor (ORF 74, KSHV GPCR) that is homologous to human chemokine receptors. KSHV GPCR is more promiscuous than most chemokine receptors in that it binds CC and CXC chemokines. For GPCRs encoded within viral genomes, KSHV GPCR is novel in that it exhibits constitutive signaling activity. It signals via the phospholipase C-inositol 1,4,5-trisphosphate-1,2-diacylglycerol pathway and activates the Jun kinase/SAP kinase and p38 MAP kinase pathways. Expression of KSHV GPCR in rat NRK fibroblasts stimulates cell proliferation. KSHV GPCR can transform mouse NIH 3T3 fibroblasts in vitro and KSHV GPCR-expressing NIH 3T3 cells form tumors in mice. Thus, KSHV GPCR displays activities of human oncogenes. Moreover, KSHV GPCR induces expression of vascular endothelial growth factor (VEGF), a potent and efficacious stimulator of angiogenesis, in NIH 3T3 cells. Thus, because of its tumorigenic and angiogenic potential, KSHV GPCR is likely to play a role in the pathogenesis of diseases associated with KSHV infection.
Discovery Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus 8) is a recently identified member of the herpesvirus family (Chang et al., 1994; Russo et al., 1996). KSHV has been found in Kaposi's sarcoma (KS) lesions of patients with AIDS and of non-AIDS-related patients (Chang et al., 1994; Moore and Chang, 1995), in normal-appearing tissue adjacent to KS lesions and in lymph nodes and peripheral blood B cells in patients with KS (Noel, 1995). KSHV has also been found in two distinct types of lymphoid proliferative disorders primary effusion lymphomas (PELs) (Cesarman et al., 1995; Arvanitakis et al., 1996) and multicentric Castleman's disease (Corbellino et al., 1996). KSHV has been shown to be a transmissible virus that infects human B cells (Mesri et al., 1996) and human endothelial cells (Flore et al., 1998). Extensive sequence analyses of fragments of KSHV have shown homology to herpesvirus saimiri and Epstein-Barr viruses (Moore et al., 1996). Since these two viruses infect and transform lymphoblastoid cells (Miller, 1974; Rangan et al., 1977), it seemed possible that KSHV may be a transforming agent also. Recently, Flore et al. (1998) showed that KSHV could transform primary human endothelial cells.
2120 Elizabeth Geras-Raaka and Marvin C. Gershengorn Lastly, it has been shown that there is seroconversion of antibodies against KSHV before the development of KS in most patients with AIDS and that KSHV appears to be a sexually transmitted disease (Gao et al., 1996a,b; Kedes et al., 1996). Thus, accumulating evidence is consistent with the idea that KSHV is involved in the pathogenesis of human primary effusion lymphomas (Nador et al., 1996) and Kaposi's sarcoma (Offermann, 1996). An open reading frame in the genome of KSHV encodes a protein that was shown to be a constitutively active G protein-coupled receptor (GPCR).
domains. On the extracellular surface is the Nterminus and three loops. Extracellular loop 1 (ECL1) connects transmembrane helix 2 (TM-2) and TM-3, ECL-2 connects TM-4 and TM-5, and ECL-3 connects TM-6 and TM-7. The cell surface membrane is spanned by seven helices. On the intracellular side are three loops and the C-terminus. Intracellular loop 1 (ICL-1) connects TM-1 and TM-2, ICL-2 connects TM-3 and TM-4, and ICL-3 connects TM-5 and TM-6. In three dimensions, the seven helices are predicted to form a helical bundle that approximates a cylinder, with TM-7 close to and interacting with TM-1 and TM-2.
Alternative names
Main activities and pathophysiological roles
KSHV GPCR; HHV 8 GPCR; IL-8-like GPCR; viral GPCR or GCR; KSHV ORF 74.
KSHV GPCR has been shown to be expressed at the mRNA level in tissues from patients with Kaposi's sarcoma and in B cell lymphomas (Cesarman et al., 1996). For GPCRs encoded within viral genomes, KSHV GPCR is novel in that it exhibits constitutive signaling activity (Arvanitakis et al., 1997). Because constitutive activation of the signaling pathways activated by KSHV GPCR induces cell proliferation
Structure The putative two-dimensional structure of KSHV GPCR is illustrated in Figure 1. Like all GPCRs, KSHV GPCR is an integral membrane protein with extracellular, transmembrane, and intracellular
Figure 1 Putative two-dimensional topology of KSHV GPCR. H2 N G C V
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Kaposi's Sarcoma-associated Herpesvirus GPCR 2121 and transformation (Post and Brown, 1996), KSHV GPCR expression transforms NIH 3T3 cells (Bais et al., 1998), and constitutively active GPCRs cause tumors in humans (Arvanitakis et al., 1998), it has been suggested that KSHV GPCR is involved in the pathogenesis of tumors associated with KSHV infection.
GENE
Accession numbers U24275; U82242; U71368; AF079845; U75698; U93872.
Sequence See Figure 2.
PROTEIN
Accession numbers 1718331; 1621029; 2246493; 1930014.
3551771;
3386561;
Q98146;
Sequence
Description of protein KSHV GPCR is a protein of 342 amino acids that appears to have the features of a GPCR including an extracellular N-terminus, three extracellular loops, seven hydrophobic, transmembrane-spanning domains, three intracellular loops, and an intracellular C-terminus (Figure 1). It is a member of the rhodopsin/ -adrenergic receptor subfamily of GPCRs but is lacking some of the amino acid residues that are highly conserved in subfamily members. For example, it has Ile in place of Asp at position 14 in TM-2, Val in place of Asp or Glu at position 24 in TM-3 (of the Asp/Glu-Arg-Tyr motif ) and Val in place of Asn at position 17 in TM-7 (of the Asn-Pro-Xaa-Xaa-Tyr motif ); numbering of positions and alignment according to Baldwin (Baldwin et al., 1997). Most importantly, KSHV GPCR exhibits marked, constitutive signaling activity (i.e. signaling in the absence of agonist) when expressed in mammalian cells (Arvanitakis et al., 1997).
Relevant homologies and species differences The amino acid sequence of KSHV GPCR shows homology to the GPCR encoded in the herpesvirus
See Figure 3. Figure 2
The nucleotide sequence of the KSHV GPCR gene.
CGTGGTGGCGCCGGACATGAAAGACTGCCTGAGGCTTTGGAAGAGACCGTACATCCT CTGCCTAAAGAGGGATCCCAGGCAGGAGTATATCAGGGGAACCACGGCGCTGTACAG TGCCTGCAGTAACGAGGTTACTGCCAGACCCACGTTTATCAACCCCCGCGTATAGCA GCTGTCCCGGATCCAGCGTCGCCTTAGCAGAGTGTCCAGTAGATTTAGTACGTGGTA AGGGAAGCAAAACACAAAAAACAGCAGCACCACAGCAACAATCACCCCCCTTACCTT CCGCCTGGCTTGCAGCTTTGTCCTCCTCACCACACACCAGGTGAGAGCATAAAACAG AATAAGGAGGGCCAGGGGTAACAGGAAACCTGCAGTAACTGACACGGTTCTGACATG CAGTCGCCAGTCTGCAGTCATGTTTCCCGCGTTCTCATAACACATGGCCTGCTTGCT GACCGGGTCGACCACCCTGCTCCTGTGTCGACAGGCATCCCCCGACAGCACCAATGC AATTAACAGTGCAGCGGATGTCAGTACCCATCCGAGGGACTGCTTCTTGGGCCAGGA ACGCGTAGAATATGCCACCAGGAGGTACCTCACTAGACTGACGCACACAACACTGAA GATATCCAAGTAGACATATAAATAGTAAAAAAAAATTTCAAGTCTGCACAAGCCTGT GGAGATGATATTGGGAAACAAAAACATCAACACTTCTGCCAATAGAGATATGCTAAG ACACAGCGAGTTTAGGCAGATACCCAGGAGCAGTATATCTATCGCTCCTGCCCGCGA TCGGTGCTTGCAAAAAATGTAGGTGACCAATCCATTTCCAAGAACATTTATGAGGAA AATCAGAGAGAGTATTCCAACGTTCCACGTGTAAGGCACCACGGTGGTCATCTCACA CACGCTCACTTCTAGGCTGAAGTTTCCAGAGTAGTCATATCCGCTCATATTTAGAGT TTCATTCCAGGATTCATCATCATCTAAGAAGATGGTTAGGAAATCCTCGGCCGCCAT
Figure 3 The amino acid sequence of KSHV GPCR. 1 51 101 151 201 251 301
MAAEDFLTIF VGILSLIFLI LAEVLMFLFP TRSWPKKQSL GNMTADWRLH GVIVAVVLLF LLQALYSAVV
LDDDESWNET NVLGNGLVTY NIISTGLCRL GWVLTSAALL VRTVSVTAGF FVFCFPYHVL PLIYSCLGSL
LNMSGYDYSG IFCKHRSRAG EIFFYYLYVY IALVLSGDAC LLPLALLILF NLLDTLLRRR FRQRMYGLFQ
NFSLEVSVCE AIDILLLGIC LDIFSVVCVS RHRSRVVDPV YALTWCVVRR WIRDSCYTRG SLRQSFMSGA
MTTVVPYTWN LNSLCLSISL LVRYLLVAYS SKQAMCYENA TKLQARRKVR LINVGLAVTS TT
2122 Elizabeth Geras-Raaka and Marvin C. Gershengorn saimiri genome (Nicholas et al., 1992; Ahuja and Murphy, 1993) and to several mammalian GPCRs (Strader et al., 1994; Power and Wells, 1996), with the highest homology to receptors for IL-8, namely CXCR1 and CXCR2 (Murphy and Tiffany, 1991; Holmes et al., 1991). The amino acid sequences deduced from several KSHV DNA isolates from KS tissues and B cell lymphomas were identical (U24275, U71368, AF079845; U75698, U93872). One amino acid sequence from a B cell lymphoma differed by one residue (U82242) and another by 13 residues in TM-4 (U82242) caused by the loss of a single nucleotide that is recovered by a downstream loss of two nucleotides.
Regulation of receptor expression Regulation of KSHV GPCR expression is not known. The levels of KSHV GPCR mRNA can be increased in lymphomatous B cells in culture by incubation with phorbol esters (Sarid et al., 1998) or butyrate (E. Cesarman, personal communication).
Release of soluble receptors There is no evidence that this occurs; it is unlikely.
SIGNAL TRANSDUCTION
Affinity for ligand(s) KSHV GPCR appears to bind a number of human CXC and CC chemokines (Arvanitakis et al., 1997). However, binding studies have been confounded by the interactions of many chemokine ligands with glycosaminoglycans. Therefore, characterization from measurements of effects of chemokines on signaling by KSHV GPCR may be more definitive than those from binding studies. In general, relative affinities of ligands for GPCRs can be estimated from relative potencies. Although most chemokines tested do not affect KSHV GPCR signaling, a small number were found that further stimulate KSHV GPCR constitutive activity (see below) and others that inhibit KSHV GPCR signaling. Human growth-related protein (GRO ) (EC50 15 nM) and IL-8 (EC50 39 nM) (Gershengorn et al., 1998) further stimulate KSHV GPCR whereas human IP-10 (EC50 39 nM) (GerasRaaka et al., 1998b), human SDF-1 (EC50 43 nM) and viral monocyte inflammatory protein II (vMIPII) (EC50 48 nM) inhibit KSHV GPCR signaling (Geras-Raaka et al., 1998a). Thus, IP-10, SDF-1, and vMIP-II are inverse agonists (or negative antagonists) of KSHV GPCR signaling.
Cell types and tissues expressing the receptor KSHV GPCR is encoded by KSHV and has been found to be expressed at the messenger RNA (mRNA) level in lesions of patients with Kaposi's sarcoma and in lymphomatous B cells (Cesarman et al., 1996).
The most important aspect of KSHV GPCR signaling is that signaling occurs in the absence of any agonist; that is, KSHV GPCR is constitutively active.
Associated or intrinsic kinases KSHV GPCR activates Jun kinase (JNK)/stressactivated protein kinase (SAP kinase) and p38 mitogen-activated protein kinase (p38 MAP kinase) but not extracellular signal-regulated kinase 2 (ERK2)/MAP kinase (Bais et al., 1998). The mechanism(s) of activation of these protein kinases is not known.
Cytoplasmic signaling cascades KSHV GPCR signals via activation of intracellular phosphoinositide-specific phospholipase C leading to formation of inositol 1,4,5-trisphosphate (IP3 ) and 1,2-diacylglycerol second messengers (Arvanitakis et al., 1997). The G protein(s) that couple KSHV GPCR to phosphoinositide-specific phospholipase C is not known. Protein kinase C is activated but calcium-dependent protein kinases have not been studied.
DOWNSTREAM GENE ACTIVATION
Transcription factors activated KSHV GPCR activates a protein kinase C-responsive promoter introduced by gene transfer (Arvanitakis
Kaposi's Sarcoma-associated Herpesvirus GPCR 2123 et al., 1997) and therefore probably acts, at least in part, via AP-1 transcription factor.
Genes induced The only specific gene that has been shown to be induced by KSHV GPCR is that for VEGF, however, other genes must be induced because KSHV GPCR transforms NIH 3T3 cells (Bais et al., 1998).
Promoter regions involved There are protein kinase C-responsive elements in the VEGF promoter.
BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY
Unique biological effects of activating the receptors KSHV GPCR exhibits properties of an oncogene in that it transforms NIH 3T3 cells; KSHV GPCRexpressing NIH 3T3 cells grow in soft agar and form tumors in nude mice (Bais et al., 1998). KSHV GPCR expression induced the expression and secretion of biologically active VEGF by NIH 3T3 cells (Bais et al., 1998).
Phenotypes of receptor knockouts and receptor overexpression mice Knockouts are not relevant as this is a virally encoded receptor. Direct overexpression in mice has not been done.
Human abnormalities KSHV GPCR is hypothesized to play a role in tumorigenesis of Kaposi's sarcoma and primary effusion lymphomas.
THERAPEUTIC UTILITY
Effects of inhibitors (antibodies) to receptors The effects of inverse agonists have not been studied in animal models.
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