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Journal of Virology, March 2004, p. 2460-2471, Vol. 78, No. 5
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.5.2460-2471.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

G Protein Selectivity Determinant Specified by a Viral Chemokine Receptor-Conserved Region in the C Tail of the Human Herpesvirus 8 G Protein-Coupled Receptor

Chaoqi Liu, Gordon Sandford, Guo Fei, and John Nicholas^*

Molecular Virology Laboratories, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, Maryland 21231

Received 2 June 2003/ Accepted 12 November 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
The viral G-protein coupled receptor (vGPCR) specified by human herpesvirus 8 (HHV-8) open reading frame 74 (ORF74) is a ligand-independent chemokine receptor that has structural and functional homologues among other characterized gammaherpesviruses and related receptors in the betaherpesviruses. Sequence comparisons of the gammaherpesvirus vGPCRs revealed a highly conserved region in the C tail, just distal to the seventh transmembrane domain. Mutagenesis of the corresponding codons of HHV-8 ORF74 was carried out to provide C-tail-altered proteins for functional analyses. By measuring receptor-activated vascular endothelial growth factor promoter induction and NF-B, mitogen-activated protein kinase, and Ca²⁺ signaling, we found that while some altered receptors showed general signaling deficiencies, others had distinguishable activation profiles, suggestive of selective G protein coupling. This was supported by the finding that vGPCR and representative functionally altered variants, vGPCR.8 (R322W) and vGPCR.15 (M325S), were affected differently by inhibitors of G_i (pertussis toxin), protein kinase C (GF109203X), and phosphatidylinositol 3-kinase (wortmannin). Consistent with the signaling data, [³⁵S]GTPS incorporation assays revealed preferential coupling of vGPCR.15 to G_q and an inability of vGPCR.8 to couple functionally to G_q. However, both variants, wild-type vGPCR, and a C-tail deletion version of the receptor were equally able to associate physically with G_q. Combined, our data demonstrate that HHV-8 vGPCR contains discrete sites of G interaction and that receptor residues in the proximal region of the cytoplasmic tail are determinants of G protein coupling specificity.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The G-protein coupled receptor (vGPCR) specified by open reading frame 74 (ORF74) of human herpesvirus 8 (HHV-8) has counterparts in all other sequenced gamma-2 herpesviruses, with the exception of alcelaphine (wildebeast) herpesvirus 1. The roles of these proteins are not understood, but they are expressed during productive (lytic) virus replication as early or early-late proteins, and it is likely, therefore, that they mediate signal transduction that results in the expression of viral and/or cellular genes that enhance viral replication. Indeed, it has been reported recently that the vGPCR of murine gammaherpesvirus 68 (MHV-68) can effect increased lytic replication in culture in the presence of agonist and that the receptor is important for lytic reactivation from latency in vivo (37, 43). It has also been established by using gene knockout recombinant viruses that vGPCRs of murine and rat cytomegaloviruses (betaherpesviruses) are important for efficient virus replication in culture and/or in vivo and that the murine cytomegalovirus M78-encoded vGPCR is a component of the virion and is necessary for efficient expression of viral immediate-early mRNA (8, 9, 19, 47). The HHV-8 vGPCR is able to activate several lytic cycle promoters in transfection assays (14). The receptor can also function as an oncogene in various experimental systems and can effect the development of Kaposi's sarcoma (KS)-like lesions in transgenic mice (6, 7, 27, 31, 73). These properties, in addition to the ability of HHV-8 vGPCR to induce vascular endothelial growth factor (VEGF) and other cytokines that may be of relevance to HHV-8 pathogenesis (13, 27, 49, 61, 65), have implicated the protein as a potential mediator of HHV-8-associated diseases such as KS, primary effusion lymphoma and multicentric Castleman's disease. Therefore, functional and mechanistic studies of HHV-8 vGPCR have been the focus of considerable research efforts, both to try to understand the basis of vGPCR-mediated transformation and to provide information that could be used to develop potentially therapeutic methods to inhibit its activity.

The activity of HHV-8 vGPCR is independent of ligand, although receptor signaling can be modulated positively and negatively by certain chemokines, including GRO (agonist) and vCCL-2 (HHV-8 ORFK4 product), SDF-1, and IP-10 (inverse agonists) (23, 24, 25, 53). G proteins that can couple functionally to vGPCR include G_q, G_i, and G₁₃ class proteins, and these can effect vGPCR-mediated activation of mitogen- and stress-activated protein kinases (ERK, p38, and JNK) and/or NF-B in a variety of cell types, including endothelial and primary effusion lymphoma cells (13, 17, 42, 45, 49, 61, 63). Thus, multiple pathways can be activated by vGPCR, and the receptor is able to couple functionally to a number of different G proteins. However, the relative contributions of different G-initiated pathways to vGPCR-effected cellular transformation and pathogenesis and to virus biology are not clear. A means of dissociating them at the level of the receptor would enable these questions to be addressed.

Structure-function studies of cellular GPCRs, primarily adrenergic and muscarinic receptors, have identified several regions of these proteins that are required for or contribute to G-protein coupling (70). The precise regions and residues involved in G-protein coupling are highly variable between different receptors, even those that are structurally closely related. Residues in all three intracellular loops (ICLs), but particularly the second and third, and also within the C tail have been implicated in at least some of the receptors investigated (2, 4, 5, 15, 18, 52, 70). In HHV-8 vGPCR-related chemokine receptor CXCR2, basic residues in the second and third ICLs, the conserved DRY motif at the start of ICL2, and C-terminal residues have been implicated in G-protein coupling (10, 12, 18, 72, 74). As for CXCR2, functional analyses of C-terminally truncated versions of CCR5 also have identified residues that are important for activity (35), but investigations of the functional importance of the most membrane-proximal regions of these or other chemokine receptors have not been reported. For some nonchemokine receptors, at least, the transmembrane-proximal region appears to be functionally significant (see, e.g., references 3, 16, 32, 56, and 69). Inhibition of signaling of such receptors by short synthetic peptides corresponding to this region indicates that the N-terminal residues of the C tail are involved directly in G coupling (39, 44, 60, 64). For the 2A/ß2-adrenergic and mGluR1/mGluR3 glutamate receptors, this region has been shown to contribute to G protein selectivity (26, 38, 50), but from these and other studies (46, 66, 68, 71), it is apparent that the C tail alone is not sufficient to determine G coupling specificity. Therefore, the transmembrane-proximal C-tail residues of cellular GPCRs can contribute to G protein coupling, and they may also contribute to G protein selectivity in some receptors.

Structural and functional studies of the HHV-8 vGPCR have identified several regions and residues of the receptor that are required for constitutive or chemokine-modulated activity (Fig. 1, upper section). Specifically, L91D and L94D substitutions in the second transmembrane domain (TM2) were found to effect loss of constitutive activity but not agonist responsiveness, ORF74-CXCR2-conserved R208 and R212 have been identified as important for interleukin-8 binding and GRO agonist activity but not for GRO binding or IP-10 suppression, and residues at the intracellular boundaries of TM2 and TM3 have been shown to modulate constitutive signaling (30, 49, 54). With regard to the last, substitutions D83A (TM2) and V142D (TM3, generating the highly conserved DRY motif of other GPCRs) effected increased constitutive signal transduction, and the latter was also found to enhance agonist responsiveness of the receptor (30, 49). The DRY motifs of other GPCRs are believed to be involved in G-protein-receptor interactions and in mediating ligand inducibility in these receptors (12, 57, 58). Other studies have determined that the N-terminal region of vGPCR is required for chemokine binding and activity and have found that the C-terminal five amino acids are essential for receptor signaling (29, 54, 61). It is notable that deletion of the C-terminal residues, rather than increasing receptor signaling as is the case for several cellular GPCRs, effects functional inhibition (61).

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FIG. 1. Structure of HHV-8 vGPCR and alignment of C-tail residues with equivalent regions of gammaherpesvirus and selected cellular chemokine receptors. The top section illustrates the predicted structure of the heptahelical receptor of HHV-8, showing residues identified experimentally as being functionally important (superscripts indicate relevant references) (see text for details). The alignment of receptor C tails shows conserved residues (boldface), relative to the HHV-8 sequence, within the membrane-proximal region (boxed). Within this region, all eight amino acids are absolutely conserved in HHV-8, HVS, and herpesvirus ateles (HVA) vGPCRs. RRV, rhesus rhadinovirus.

We have undertaken structure-function studies of HHV-8 vGPCR to identify cytoplasmic residues that are important for constitutive activity, residues that may interact with G proteins and be appropriate targets for vGPCR inhibitors. Comparison of the vGPCRs of HHV-8, herpesvirus saimiri (HVS), and other gamma-2 herpesviruses revealed the presence of an 8-amino-acid conserved motif, GSLFRQRM, in the cytoplasmic tails of the receptors (Fig. 1, lower section), equivalents of some of which have been noted previously to be highly conserved in cellular GPCRs. By undertaking site-directed mutagenesis of HHV-8 ORF74 to introduce amino acid substitutions within this conserved region and functional analyses of the encoded proteins, we found that at least one substitution at each of five targeted positions perturbed vGPCR signaling. General, ERK-specific, and NF-B-specific signaling effects were position and substitution dependent. Representative variant vGPCRs that were selectively able to activate ERK (M325S) or NF-B (R322W) signaling were analyzed further to investigate signaling pathway activation, their physical interactions with G proteins, and their functional coupling to G_q and G_i proteins. Combined, the presented data identify residues within the HHV-8 vGPCR cytoplasmic tail that, while not necessary for G association with receptor, are important for G protein coupling and selectivity; equivalent residues in other viral and cellular chemokine receptors may function similarly.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tissue culture and transfections. HEK293 cells were grown in Dulbecco modified Eagle's medium supplemented with 5% fetal calf serum and antibiotics (penicillin and streptomycin). Transient transfections of HEK293 cells were undertaken in six-well plates by standard calcium phosphate precipitation, with precipitates applied for 8 to 15 h prior to changing the medium for further incubation (48 h) prior to cell harvesting for determinations of intracellular calcium concentration (see below), luciferase activity, or levels of active (phosphorylated) ERK. Total amounts of DNA transfected in the various experiments were between 2 and 4 µg (held constant between samples in each experiment by addition of appropriate empty vector DNA).

Plasmids and mutagenesis. Site-directed mutagenesis of the vGPCR open reading frame was carried out on a uracil-containing M13-vGPCR template by the Kunkel procedure (36). Wild-type or specifically altered vGPCR ORF sequences were cloned as EcoRI fragments into either pSG5 (Stratagene, La Jolla, Calif.) or pJH405 (pSG5-based vector containing human immunoglobulin IgG Fc-coding sequences; a gift from Diane Hayward) to enable expression in transfected eukaryotic cells of the native or C-terminally Fc-extended vGPCR proteins. G_q-coding sequences were amplified by PCR from a human cDNA library and cloned (as an EcoRI fragment) in frame with Flag epitope-coding sequences in the pSG5-based vector pJH492 (provided by Diane Hayward). ERK1-coding sequences were derived by PCR amplification from a human cDNA library and cloned into a green fluorescent protein (GFP) ORF-containing pSG5-based vector (pJH521, provided by Diane Hayward) to produce an ERK1-GFP fusion protein in transfected cells. Luciferase reporter pVEGF(1176)-Luc, containing full-length VEGF promoter sequences upstream of the luciferase ORF, has been described previously (40) and was kindly provided by Gilles Pagès. A luciferase reporter containing three upstream NF-B sites was generated by cloning appropriate double-stranded oligonucleotides into the NheI site of pGL2-basic (Promega, Madison, Wis.) containing herpes simplex virus thymidine kinase promoter sequences (55) (a gift from Steve Baylin). The NF-B-binding oligonucleotide sequence was CTAGCGAGGGGACTTTCCCAGG. The pcDNA3-based plasmid pY-CAM-2 contains cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) ORFs linked by coding sequences for phage coat protein M13 and calmodulin (41) and was kindly provided by Roger Tsien.

Western blotting, immunofluorescence, coprecipitation, and immunoprecipitation. For ERK activation assays, HEK293 cells transfected with pSG5-vGPCR (wild type or variants) or pSG5 (negative control) were harvested and disrupted in radioimmunoprecipitation assay buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40) containing 100 mM ammonium sulfate, 1 mM phenylmethylsulfonyl fluoride, 100 µM sodium orthovanadate, 50 mM sodium fluoride, and 25 µg (each) of aprotinin and leupeptin per ml. For vGPCR-Fc extractions and vGPCR-Fc-G_q coprecipitation assays, cells were disrupted in lysis buffer (20 mM Tris-HCl [pH 7.5], 1% Cymal-5 [Anatrace Inc., Maumee, Ohio], 100 mM ammonium sulfate, 1 mM phenylmethylsulfonyl fluoride, 100 µM sodium orthovanadate, 50 mM sodium fluoride, and 25 µg [each] of aprotinin and leupeptin per ml) for 30 min at 4°C. Extracts were clarified by centrifugation (14,000 x g, 30 min). Coprecipitation assays were performed by addition of protein A-agarose to cell extracts, incubation at 4°C overnight (on a rocker), and subsequent washing of protein A-bound complexes by repeated sedimentation and resuspension in fresh lysis buffer. Prior to gel loading, protein samples were heated at 100°C for 5 min, or at 55°C for 1 h for vGPCR-Fc-containing samples, in denaturation buffer. Size-fractionated proteins were electrophoretically transferred to nitrocellulose membranes for Western analysis. Antibodies to tyrosine-phosphorylated (active) ERK and total ERK were obtained from Santa Cruz Biotech (Santa Cruz, Calif.) (catalog numbers sc-7383 and sc-94G, respectively), and anti-Flag antibody (to detect G_q-Flag) was obtained from Sigma (St. Louis, Mo.) (catalog number 3165). Horseradish peroxidase-conjugated anti-goat IgG (Santa Cruz Biotech; catalog number sc-2020) or anti-rabbit IgG (Sigma; catalog number A-9169) secondary antibodies were used for visualization of membrane-bound proteins. Rabbit antiserum directed against a peptide corresponding to vGPCR N-terminal residues 4 to 16 has been described previously (14) and was provided by Gary Hayward. Immunofluorescence assays to confirm appropriate expression of vGPCR variants were undertaken after methanol fixing of cells and application of vGPCR-terminal antiserum and fluorescein isothiocyanate-conjugated anti-rabbit IgG detection antibody (Santa Cruz Biotech; catalog number sc-2012).

¹²⁵I-GRO binding assays. HEK293 cells (in six-well plates) were transfected with wild-type or variant vGPCR expression plasmids. After 48 h, the medium was removed and replaced with 0.5 ml of ice-cold binding buffer (50 mM HEPES, 1 mM CaCl₂, 5 mM MgCl₂, 0.5% bovine serum albumin [pH 7.4]) containing 20 nCi (10 pmol) of ¹²⁵I-GRO (Amersham; catalog number IM305), and incubation was carried out at 4°C for 2 h. The binding buffer and unbound ¹²⁵I-GRO were removed, and the cell monolayers were rinsed rapidly with four 1-ml washes of binding buffer supplemented with 0.5 M NaCl. Cells were harvested into 1 ml of binding buffer and recovered by centrifugation, and cell-associated radiation was quantified in a gamma-radiation counter. Parallel assays were carried out on pSG5-transfected cells to determine background levels of nonspecific or cell-receptor-associated ¹²⁵I-GRO binding.

Calcium signaling assays. Differences in the intracellular calcium concentrations in cells expressing and not expressing vGPCR were determined by fluorometric analysis with calcium-responsive Y-CAM-2 protein (41) to measure fluorescence resonance energy transfer (FRET) from the CFP to YFP domains of Y-CAM-2. Increased FRET indicates increased calcium concentration. An Aminco Bowman-2 spectrofluorometer was used to obtain emission scans (450 to 550 nm) from cells transfected with pY-CAM-2 and pSG5-vGPCR or pSG5 (negative control) plasmids; an excitation wavelength of 410 nm was used to excite CFP specifically such that YFP-derived emission was dependent on FRET. CFP-specific emission peaks (475 nm) from different cell samples were set at 60% maximum range, thereby normalizing any variations in transfection efficiency.

Signaling assays with kinase and G_i inhibitors. Pertussis toxin (PTx), GF109203X, wortmannin, and PD98059 (all obtained from Calbiochem, San Diego, Calif.) were used to assess the utilization of G_i, protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K), and mitogen-activated protein kinase (MAPK)/ERK kinase (MEK), respectively, in vGPCR-mediated signal transduction. In these experiments, cells were treated for either 16 h (PTx) or 2 h with PTx, GF109203X, wortmannin, and PD98059 at 50 ng/ml, 3 µM, 25 nM, and 20 µM, respectively, prior to cell harvest for preparation extracts.

G_q protein activation assays. [³⁵S]GTPS labeling of G proteins in cell membrane preparations and immunoprecipitation of labeled G_q/11 were done essentially as described previously (1, 21, 62). Cells transfected with expression plasmids for vGPCR, vGPCR variant 8 or 15, or pSG5 (negative control) were lysed by Dounce homogenization (using a disposable microcentrifuge tube Dounce homogenizer) in buffer A (10 mM HEPES, 10 mM EDTA [pH 7.4]), and membranes were pelleted at 16,000 x g in a microcentrifuge. The pellets were resuspended in buffer B (10 mM HEPES, 0.1 mM EDTA [pH 7.4]), homogenized by Dounce homogenization, and repelleted by centrifugation. Membrane pellets were then resuspended in assay buffer (10 mM HEPES, 100 mM NaCl, 10 mM MgCl₂ [pH 7.4]) to a protein concentration of 2 mg/ml. Assays were carried out by adding 1 µCi of [³⁵S]GTPS (1000 Ci/mmol) and GDP (to a 10 µM final concentration) to 50-µl aliquots of membranes and incubating at 30°C for 10 min. Reactions were stopped by addition of 1 ml of ice-cold assay buffer, membranes were pelleted by centrifugation at 16,000 x g, and pellets were disrupted in solubilization buffer (100 mM Tris-HCl, 20 mM NaCl, 1 mM EDTA, 1.25% NP-40 [pH 7.4]) containing 0.2% sodium dodecyl sulfate. An equal volume of solubilization buffer (without sodium dodecyl sulfate) was then added, and the supernatants were cleared by centrifugation. For immunoprecipitations of G_q/11 proteins, supernatants were incubated at 4°C with G_q/11 antibody (Santa Cruz Biotech; catalog number sc-392) for 4 h before addition of protein A-agarose and overnight incubation at 4°C. Immune complexes were then sedimented and washed by repeated cycles of centrifugation and additions of fresh solubilization buffer. The radioactivity in the washed pellets was quantified by liquid scintillation spectrometry.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Site-directed mutagenesis of gammaherpesvirus-conserved vGPCR residues. The vGPCRs specified by HHV-8 and HVS are homologous but diverged proteins, sharing 36% amino acid identity. Within each of the vGPCR C tails, immediately after the last transmembrane domain, is a run of eight conserved residues, GSLFRQRM (residues 318 to 325 of HHV-8 vGPCR), which we noticed were identical in the HHV-8 and HVS vGPCRs and identical or highly conserved in other gamma-2 herpesvirus vGPCRs; some of these residues are conserved in other chemokine receptors (Fig. 1). The remainder of the C tails of the vGPCRs, and vGPCRs versus cellular GPCRs, are divergent. This prompted us to undertake mutational analysis of HHV-8 vGPCR within the conserved motif and to investigate the signaling activities of the altered proteins to determine the functional importance of these residues. Codons 320 to 325 were targeted for mutagenesis with degenerate mutagenic oligonucleotides to generate random mutations at each position. The vGPCR variants that were obtained and selected for functional analysis (see below) are listed in Table 1.

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TABLE 1. Substitution mutations introduced into the vGPCR C tail

Expression of vGPCR variants. Each altered ORF was cloned into the eukaryotic expression vector pSG5, and expression of each of the proteins in transfected HEK293 cells was checked by immunofluorescence microscopy with vGPCR antiserum directed to the N-terminal region (14) (Fig. 2A). Each of the proteins appeared to be expressed appropriately, at the cell membrane, and at similar levels. No significant staining of pSG5-transfected cells was detected. To quantify their expression, ¹²⁵I-GRO binding to vGPCR vector-transfected cells was measured; these data (Fig. 2B) confirmed that each of the altered receptors, with the exception of variant 7 (R322V), was expressed at the cell surface at a level similar to (at least 65%) that of wild-type vGPCR. Expression of a selection of the altered receptors (including functionally impaired variants [see below]) relative to wild-type vGPCR was checked also by Western blotting. These proteins were expressed as Fc fusions that were precipitated from cell lysates with protein A-agarose, prior to gel electrophoresis and blotting, to remove background staining that was seen in total cell extracts (not shown). Wild-type and variant proteins were found to be expressed at similar levels (Fig. 2C).

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FIG. 2. Expression of vGPCR variants in transfected HEK293 cells. (A) Immunofluorescence assays using rabbit antiserum directed to the N-terminal region of vGPCR (14) for the detection of wild-type (WT) and variant receptors in transfected cells. Similar cell surface fluorescence was seen for all variants, with no significant staining of pSG5 (empty vector)-transfected cells. (B) 125I-GRO binding assays (see Materials and Methods) to measure surface expression of the wild-type and engineered vGPCRs. Data were derived from five independent transfections for each receptor and are expressed relative to 125I-GRO binding by wild-type vGPCR (100%). Results are shown as means ± standard deviations. Background binding (to pSG5-transfected cells) has been subtracted from the data presented. (C) Western blotting for the detection of vGPCR-Fc fusion proteins. Those tested showed expression levels equivalent to that of wild-type vGPCR.

Signaling by native and altered vGPCR proteins in VEGF promoter and NF-B reporter assays. As vGPCR has been demonstrated previously to induce VEGF and to activate NF-B (7, 13, 42, 49, 63, 65, 73), we tested the effects of the introduced amino acid substitutions on vGPCR activity by using luciferase-linked VEGF promoter and NF-B binding site reporters (see Materials and Methods) together with the vGPCR expression vectors in transfection assays with HEK293 cells. Both reporters were activated as a function of vGPCR, above the levels obtained with the empty expression vector, pSG5 (Fig. 3). The activities of several of the vGPCR variants were substantially reduced with respect to those of either one or both of the reporters; the different relative levels of activation of each of the reporters compared to the levels seen with wild-type vGPCR suggested that some of the variants were altered in their abilities to signal via one or more of multiple pathways activated by the native vGPCR. Substitutions at positions 320 (L to R), 321 (F to L), and 323 (Q to P) significantly reduced or abolished activation of both reporters, while other substitutions, most notably R322V (variant 7), R322W (variant 8), and M325S (variant 15), resulted in selective activation of one of the reporters. This selectivity in reporter activation suggested that mutation of these C-tail residues may selectively affect the functional association of different subclasses of G proteins with vGPCR.

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FIG. 3. Activation of VEGF promoter and NF-B reporters by wild-type (WT) and variant vGPCRs. HEK293 cells were cotransfected with pVEGF(1176)-Luc (40) or NF-B-Luc (see Materials and Methods) and each of the vGPCR expression vectors or pSG5 (negative control). After 48 h, luciferase activities in cell extracts were determined. Data were derived from four independent experiments and normalized to activities obtained for wild-type vGPCR (set at 100%). Values shown are means ± standard deviations.

Correlation of ERK and VEGF promoter activation by vGPCR variants. To investigate the basis of the differences in levels of activation of the VEGF promoter by wild-type and variant vGPCRs, we undertook experiments to determine the effects of vGPCR and variants 8 (R322W) and 15 (M325S) on activation of ERK, an activator of HIF-1 that is the major effector of VEGF transcriptional induction (40). For these experiments, we used an ERK1-GFP expression construction, pSG5-ERK1GFP, to allow the expression of the fusion protein in cells cotransfected with pSG5-vGPCR and thereby enable measurement of ERK1 activation (phosphorylation) specifically in transfected cells. Analysis of transfected HEK293 cell extracts by Western blotting for the detection of phosphorylated ERK showed that the ERK1-GFP fusion protein was activated as a function of vGPCR expression (Fig. 4A). ERK1-GFP activation mirrored the vGPCR-induced phosphorylation of endogenous ERK1 and ERK2, detectable in this experiment, demonstrating the validity of using ERK1-GFP as a reporter for MAPK signaling in cells coexpressing vGPCR. Levels of ERK and ERK1-GFP independent of phosphorylation status were essentially the same in all samples, as determined by using a phosphorylation-independent ERK antibody.

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FIG. 4. Activation of ERK by vGPCR and signaling-altered variants 8 (R322W) and 15 (M325S). (A) HEK293 cells were transfected with pSG5 (vector) or pSG5-vGPCR, either with or without cotransfected pSG5-ERK1GFP. Cell extracts were analyzed by Western blotting for the presence of tyrosine-phosphorylated ERK (top panel) or total ERK. (B) ERK1-GFP activation as a function of vGPCR, vGPCR.8, and vGPCR.15 expression in transfected HEK293 cells. WT, wild type.

We then tested the abilities of vGPCR.8 (R322W; NF-B-Luc positive, VEGF-Luc negative) and vGPCR.15 (M325S; VEGF-Luc positive, NF-B-Luc decreased) to activate ERK. The results are shown in Fig. 4B. While vGPCR.15 was able to mediate ERK signaling, no ERK activation by vGPCR.8 was detected. Thus, activation of ERK by these variants, and by wild-type vGPCR, mirrors the activities seen in the VEGF promoter-luciferase assays and suggests that vGPCR.8 may be unable to couple efficiently to G_q, which can initiate MAPK signaling via direct activation of phospholipase C (PLC).

Pathways of ERK activation by vGPCR. To investigate the utilization of G proteins and signal transduction pathways for vGPCR-mediated activation of ERK, we undertook ERK activation assays in the absence or presence of the PKC and G_i inhibitors GF109203X and PTx. The main GPCR-activated signaling pathways leading to ERK activation are indicated in Fig. 5A. As before, cotransfection of vGPCR and ERK1-GFP expression plasmids into HEK293 cells led to strong activation of ERK in the transfected cells, with no detectable induction of phosphorylated ERK1-GFP in the pSG5-transfected cells (negative control) (Fig. 5B). Addition of the PKC inhibitor GF109203X completely blocked vGPCR-mediated ERK activation; levels were equivalent to those obtained in the presence of the MEK (ERK kinase) inhibitor PD98059. These data show that vGPCR signaling to ERK is mediated via PKC, consistent with the idea that G_q is the major effector of such signaling.

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FIG. 5. Pathway of vGPCR-mediated ERK activation. (A) Pathways leading to ERK activation by G protein-coupled receptors. Boldface arrows indicate major signaling pathways. (B) Role of PKC in vGPCR activation of ERK. PKC and MEK inhibitors GF109203X (GF) and PD98059 (PD) were added to vGPCR- and ERK1-GFP-coexpressing HEK293 cells for and left for 2 h prior to harvesting of the cells for protein extraction and Western analysis for the detection of phosphorylated (activated) and total ERK1-GFP. (C) Gi independence of ERK activation by vGPCR. Receptor- and ERK1-GFP-cotransfected cells were either untreated or treated with PTx (Gi inhibitor) for 16 h prior to the preparation of cell extracts for Western analysis. (Conditions used for PTx treatment were identical to those found to inhibit NF-B signaling by vGPCR.8 [see Fig. 7A]). WT, wild type.

To exclude the possibility of G_i involvement in vGPCR-activated ERK signaling (via actions of released Gß), we undertook analogous experiments in the absence or presence of PTx. In these assays, we included vGPCR.8 and vGPCR.15 in addition to wild-type vGPCR. PTx had no significant effect on either of the receptor variants (in this experiment, vGPCR.8 effected detectable, but reduced, ERK activation), indicating that vGPCR signaling to ERK is independent of G_i (Fig. 5C). These data, together with those of Fig. 5B, suggest that vGPCR.15 (M325S) is predominantly G_q coupled and that vGPCR.8 (R322W) is unable to couple effectively to G_q.

Calcium signaling by vGPCR, vGPCR.8, and vGPCR.15. To provide further support for the hypothesis of distinct G_q coupling abilities of vGPCR.8 and vGPCR.15, relative to each other and to wild-type vGPCR, we next tested the abilities of these receptors to effect calcium signaling. Mobilization of calcium by GPCR signaling is mediated primarily by G_q activation of PLC to effect inositol-1,4,5-triphosphate accumulation and release of ER-stored calcium (Fig. 5A). As vGPCR is constitutively active, we used a plasmid-encoded calcium-responsive reporter to detect receptor-effected changes in steady-state average intracellular calcium within the receptor-transfected cell population. We cotransfected each of the vGPCRs, or pSG5 (negative control), with pY-CAM-2, a plasmid expressing a protein comprising calmodulin-linked CFP and YFP that responds conformationally to changes in calcium concentration over physiological ranges (41). The conformational change can be monitored with a spectrofluorometer by measuring CFP-to-YFP FRET following specific excitation of CFP at an appropriate wavelength (e.g., 410 nm) (Fig. 6A). A representative example of one of multiple experiments using vGPCR, vGPCR.8, and vGPCR.15 is shown in Fig. 6B. Variant 15 was consistently more active than wild-type vGPCR in this assay, indicating preferential G_q coupling as a consequence of the M325S change, while calcium concentrations in cells transfected with variant 8 (R322W) were reduced below the basal levels seen in the pSG5-transfected cells. The latter result is consistent with G_i-mediated inhibition of calcium release, which has been shown for the somatostatin receptor (34) and suggested for vGPCR on the basis of its ability to inhibit PLC activity in a G_i-dependent manner (17), and indicates preferential G_i coupling by vGPCR.8.

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FIG. 6. Calcium signaling by vGPCR, vGPCR.8, and vGPCR.15. (A) Diagrammatic representation of Y-CAM-2 (41) and its use to detect [Ca2+]-dependent FRET. (B) The Y-CAM-2 expression plasmid was cotransfected into HEK293 cells with vGPCR, vGPCR.8, or vGPCR.15 expression vector (or pSG5 [negative control]) for FRET-based calcium mobilization assays. Excitation of CFP in transfected cells was effected by using a wavelength of 410 nm; emission wavelength shifts from 475 nm (CFP emission peak) to 527 nm (YFP emission peak) result from increased FRET due to Ca2+-induced conformational change in the CFP-calmodulin-YFP reporter encoded by pY-CAM-2. Representative scans from one of multiple experiments are shown. WT, wild type.

Pathways of NF-B activation by vGPCR, vGPCR.8, and vGPCR.15. Reported data indicate that vGPCR can mediate NF-B signaling via G_i, G_q, and G₁₃ proteins (13, 17, 42, 63), with the balance of G subtype and pathways utilized being cell type determined. To identify the pathways of NF-B activation by vGPCR, vGPCR.8 (R322W), and vGPCR.15 (M325S) in HEK293 cells, and to distinguish between their couplings to different classes of G proteins, we undertook NF-B reporter assays in the absence or presence of inhibitors of PKC (GF109203X), PI3K (wortmannin), and G_i (PTx). The results of these experiments are shown in Fig. 7A. The data reveal distinct inhibition profiles of the chemical agents on the three receptors. PTx effected almost complete inhibition of vGPCR.8 activity, while inhibiting vGPCR.15 activity by only 25%, demonstrating predominant G_i coupling by the R322W variant and weak coupling to G_i by vGPCR.15. GF09203X had only small (25%) inhibitory effects on NF-B signaling by vGPCR.15, demonstrating minor involvement of PKC, whereas wortmannin reduced signaling by vGPCR.15 by 75%, implicating Gß and/or Pyk2 activation of PI3K as the major route(s) of NF-B activation (Fig. 7B). For vGPCR.8, inhibition of PKC and PI3K decreased NF-B-Luc activation to approximately 60 and 15% of that obtained in the absence of drug, demonstrating the involvement of PKC and central importance of PI3K in NF-B activation by this receptor variant. The lack of inhibition of wild-type vGPCR by PTx, GF109203X, or wortmannin is consistent with previous reports documenting the utilization of multiple G proteins and pathways for NF-B activation by this receptor; thus, inhibition of one pathway can presumably be compensated for by signaling to NF-B through others (Fig. 7B). Combined, our data demonstrate that vGPCR.8 (R322W) is almost exclusively G_i coupled, that vGPCR.15 (M325S) is very weakly coupled to G_i, and that Gß activation of PI3K represents the major route of NF-B activation.

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FIG. 7. NF-B activation pathways utilized by vGPCR, vGPCR.8, and vGPCR.15. (A) NF-B-Luc reporter assays of extracts derived from HEK293 cells transfected with expression vector for vGPCR, vGPCR.8 (R322W), or vGPCR.15 (M325S) in the presence or absence of Gi, PKC, or PI3K inhibitors (PTx, GF109203X [GF], or wortmannin [Wt], respectively). Receptor variant 8 shows Gi dependence for NF-B activation, whereas vGPCR.15 can effect NF-B signaling through other routes that involve, to different degrees, the activities of PKC and PI3K. The chemical agents did not inhibit NF-B signaling by wild-type vGPCR. The data presented are from triplicate transfections and are expressed as mean percent inhibition (relative to GPCR signaling in the absence of drug) ± standard deviation. (B) GPCR-activated signaling pathways leading to NF-B activation and those (boldface arrows) that our data indicate are the main pathways utilized by vGPCR (Gi, Gq, and G12/13 coupled), vGPCR.8 (Gi coupled), and vGPCR.15 (Gq coupled [see Fig. 9]) in HEK293 cells. Inhibitory activities of the reagents used in our experiments are indicated by the grey bars.

Interactions of vGPCR, vGPCR.8, and vGPCR.15 with G_q. While our data demonstrated preferential G_i coupling by vGPCR.8 and strongly suggested predominant G_q coupling by vGPCR.15, we wanted to demonstrate the differential G_q coupling by vGPCR, vGPCR.8, and vGPCR.15. Therefore, we undertook experiments to determine whether these receptors showed differences in their abilities to interact with G_q. In these experiments, receptor-Fc fusion proteins and Flag-tagged G_q were coexpressed in transfected HEK293 cells, cell extracts were made, and receptor-bound G_q was identified in protein A-agarose-precipitated material by Western analysis. In addition to vGPCR, vGPCR.8, and vGPCR.15, we also used a C-terminally truncated receptor fused to Fc (Fig. 8A). The results of the coprecipitation assays revealed that the R322W and M325S mutations in vGPCR.8 and vGPCR.15 had no effect on G_q-Flag binding; indeed, deletion of the C tail also had no detectable effect on vGPCR-G_q association. That these interactions were specific and representative of true vGPCR-G interactions, requiring appropriate conformation of the receptor, was indicated by the failure of variants containing substitutions of conserved, structural C residues (Fig. 8A) to coprecipitate G_q (Fig. 8C). Therefore, these data demonstrate the existence of a high-affinity G-binding interface distinct from the C-tail sequences.

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FIG. 8. Gq-vGPCR association. Binding of Gq to vGPCR and variants 8 (R322W) and 15 (M325S) and a C-terminally deleted version of vGPCR (lacking amino acids after G318) was examined by using coprecipitation assays with receptor-Fc fusion proteins and Flag-tagged Gq. Proteins were expressed in cotransfected cells and precipitated from cell extracts by using protein A-agarose. (A) Diagrammatic representation of the vGPCR-Fc fusion proteins made, including cysteine variants 22 (C39A), 23 (C118A), and 24 (C196A). (B) Coprecipitation assays with vGPCR C-tail variants; the ability of vGPCRC to bind Gq-Flag demonstrates the existence of a C-tail-independent interaction interface on the receptor. WT, wild type. (C) Coprecipitation assays with C-to-A variants, demonstrating specificity of the assay and dependence on conformational integrity (requiring intramolecular disulfide bridging) of vGPCR for Gq binding.

G_q coupling by wild-type and variant vGPCRs. We next turned to a functional approach to provide direct evidence for preferential G_q coupling by vGPCR.15 relative to vGPCR.8 and vGPCR. We utilized [³⁵S]GTPS, an unhydrolyzable G substrate, and an antibody specific for G_q and G₁₁ to coprecipitate these G_q proteins from vGPCR-expressing membrane preparations from transfected cells following incubation to label receptor-activated G proteins. The sedimented radiation in washed immunoprecipitates was quantified by scintillation counting. The results (Fig. 9) confirmed that vGPCR and vGPCR.15 were able to couple functionally with G_q proteins, with the latter effecting higher levels of G_q and G₁₁ labeling. In contrast, immunoprecipitated counts for vGPCR.8 were essentially indistinguishable from those obtained for the negative control (transfected pSG5); the counts obtained represent background levels (with nonspecific, protein A-agarose-associated counts subtracted) and most probably reflect activities of endogenous GPCRs. The data from these experiments demonstrate that vGPCR.15 (M325S) is preferentially G_q coupled and confirm that vGPCR.8 (R322W) is unable to associate functionally with G_q.

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FIG. 9. Gq protein coupling by vGPCR, vGPCR.8, and vGPCR.15. Receptor-coupled and activated G proteins in HEK293 cells transfected with expression vector for vGPCR, vGPCR.8, or vGPCR.15, or with pSG5 (negative control), were labeled by addition of [35S]GTPS to membrane fractions derived from these cells. The relative abilities of the different receptors to couple to Gq and G11 were determined by immunoprecipitation of these G proteins (with an antibody recognizing both), followed by gamma radiation counting of the precipitated material. The results shown are derived from triplicate experiments. Nonspecific background counts, present in protein A-agarose precipitates in the absence of antibody, have been subtracted from the presented data; values shown are means ± standard deviations.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The data presented in this report show for the first time that amino acid residues within the transmembrane-proximal region of the C tail of HHV-8 vGPCR are functionally important and involved in G protein coupling and selectivity. These residues are generally conserved in other gamma-2 herpesvirus vGPCRs, and either R or K (basic) residues are found in cellular chemokine receptors at a position equivalent to HHV-8 vGPCR R322, which we have shown is important for G_q coupling. These conserved residues may be functionally analogous in at least some of these other chemokine receptors.

Our data indicate that for the gamma-2 herpesvirus ORF74-type chemokine receptors, the proximal portions of the cytoplasmic tails may be key determinants of G protein interactions. From the elegant studies of Schwarz and Murphy (61), it is known that the C-terminal five amino acids of vGPCR are essential for vGPCR-mediated NF-B and AP-1 activation and may also be important for interactions of the receptor with G proteins. These results contrast with those obtained from studies of various cellular GPCRs, which indicate that the transmembrane-distal regions of the C tails are not required for signal transduction but rather are often associated with suppression of G protein activation, possibly by masking one or more G:receptor interaction interfaces or by increasing receptor internalization (see, e.g., references 28, 32, 33, 48, and 59). In the case of the US28-encoded vGPCR of human cytomegalovirus, a region (residues 317 to 355) of the C tail (residues 295 to 355) has been demonstrated to be involved in receptor internalization (67). Whatever the precise roles of the membrane-proximal and C-terminal residues of HHV-8 vGPCR are in G interaction, our coprecipitation data (Fig. 8) show that there is at least one other region of the receptor that can interact with G proteins, as a C-terminally deleted vGPCR can associate stably with G_q. As residues within ICL2 and ICL3 have been reported to be important for chemokine-induced signaling by some cellular chemokine receptors, such as CCR2, CXCR4, and vGPCR-related CXCR2 (4, 12, 18, 52, 72, 74), it is likely that ICL2 and/or ICL3 residues of vGPCR also interact with G.

One significant aspect of the present study is that it has identified a target within vGPCR for potentially therapeutic inhibitory reagents. The viral receptor has been implicated as a contributory factor in HHV-8 pathogenesis, particularly KS, where paracrine mechanisms, probably involving induced VEGF and other secreted factors, are key (6, 7, 20, 49, 73). Furthermore, vGPCR is likely to be involved in the activation of viral gene expression during lytic replication (13, 14), and recent in vivo data from experiments utilizing MHV-68 vGPCR-disrupted virus indicate that such activity may be particularly important for viral reactivation, that is, for activation of lytic gene expression in cells that are not normally supportive of lytic replication (37, 43). Therefore, targeting HHV-8 vGPCR with inhibitory agents directed to uncoupling of receptor-G protein interactions may be an effective antiviral strategy. Another aspect of the results presented here is that by dissociating G_i from G_q signal transduction through HHV-8 vGPCR mutagenesis, reagents have been generated to allow the determination of the relative importance of these vGPCR-activated signal transduction pathways to cellular and viral gene expression, viral replication, and viral pathogenesis. Thus, by substituting HHV-8 and MHV-68 vGPCRs with HHV-8 vGPCR.8 (R322W) and vGPCR.15 (M325S) or equivalent MHV-68 vGPCR variants, it should be possible to determine the relative contributions of G_i- and G_q-initiated pathways to viral replication and reactivation in vitro (HHV-8 and MHV-68) and in vivo (MHV-68). In light of the reported chemokine-dependent pathogenic effects of HHV-8 vGPCR in transgenic mice (31) and agonist-dependent vGPCR-mediated enhancement of MHV-68 replication in culture (37), our reagents may help determine whether the molecular basis of these phenomena relates to agonist-induced differences in G protein utilization. There is precedent for such effects, that is, ligand-induced receptor conformational changes leading to distinct G protein coupling, among cellular ligands and receptors (11, 22, 51).

In conclusion, we have presented data identifying the proximal region of the C tail of HHV-8 vGPCR as critically important for G-protein coupling and have generated vGPCR variants that display G protein selectivity. These data and reagents are relevant to the design of potentially therapeutic vGPCR-inhibitory reagents and for future investigations of the relative contributions of different vGPCR-activated signaling pathways to viral replication, reactivation, and pathogenesis.

 
We are grateful to Roger Tsien, Gilles Pagès, Gary Hayward, and Diane Hayward for provision of plasmid and immunological reagents that were used in this study.

This work was supported by NIH grant CA76445.

 
* Corresponding author. Mailing address: Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, 1650 Orleans St., Room 309, Baltimore, MD 21231. Phone: (410) 502-6801. Fax: (410) 502-6802. E-mail: nichojo{at}jhmi.edu.

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Journal of Virology, March 2004, p. 2460-2471, Vol. 78, No. 5
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.5.2460-2471.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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