Mutational analysis of the R33-encoded G protein-coupled receptor of rat cytomegalovirus: identification of amino acid residues critical for cellular localization and ligand-independent signalling

Yvonne K. Gruijthuijsen1, Erik V. H. Beuken1, Martine J. Smit2, Rob Leurs2, Cathrien A. Bruggeman1 and Cornelis Vink1

1 Department of Medical Microbiology, Cardiovascular Research Institute Maastricht, University of Maastricht, PO Box 5800, 6202 AZ Maastricht, The Netherlands
2 Division of Medicinal Chemistry, Leiden/Amsterdam Centre for Drug Research, Free University, 1081 HV Amsterdam, The Netherlands

Correspondence
Cornelis Vink
kvi{at}lmib.azm.nl


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The rat cytomegalovirus (RCMV) R33 gene encodes a G protein-coupled receptor (GPCR), pR33, which possesses agonist-independent, constitutive signalling activity. To characterize this activity further, we generated a series of point and deletion mutants of pR33. Both expression of and signalling by the mutants was evaluated. Several point mutants were generated that contained modifications in the NRY motif. This motif, at aa 130–132 of pR33, is the counterpart of the common DRY motif of GPCRs, which is known to be involved in G protein coupling. We found that mutation of the asparagine residue within the NRY motif of pR33 (N130) to aspartic acid resulted in a mutant (N130D) with similar signalling characteristics to the wild-type (WT) protein, indicating that N130 is not the determinant of constitutive activity of pR33. Interestingly, a mutant carrying an alanine at aa 130 (N130A) was severely impaired in Gq/11-mediated, constitutive activation of phospholipase C, whereas it displayed similar levels of activity to pR33 in Gi/0-mediated signalling. Another protein that contained a modified NRY motif, R131A, did not show constitutive activity, whereas mutants Y132F and Y132A displayed similar activities to the WT receptor. This indicated that residue R131 is critical for pR33 function in vitro, whereas Y132 is not. Finally, we identified two consecutive arginines within the C-terminal tails of both pR33 and its homologue from human CMV, pUL33, which are important for correct cell-surface expression of these receptors.


   INTRODUCTION
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INTRODUCTION
METHODS
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DISCUSSION
REFERENCES
 
Cytomegaloviruses (CMVs) are species-specific betaherpesviruses that cause acute, persistent and latent infections in both humans and animals. The ability of CMVs to induce lifelong latent infections implies that these viruses are highly adapted to their hosts. Specifically, they employ a wide array of sophisticated strategies aimed at subversion of the host's antiviral defence mechanisms. Among the CMV genes that are thought to be involved in these strategies are those encoding homologues of crucial immune effector or regulatory proteins of the host, such as G protein-coupled receptors (GPCRs).

GPCRs form a large and diverse family of receptors that function in signal transduction across cell membranes. They are composed of a central core of seven transmembrane (7-TM) helices connected by three intracellular and three extracellular loops. The majority of these receptors activate G proteins and thereby transduce a wide variety of extracellular messages into intracellular responses. Within the genomes of all sequenced CMVs, genes have been identified that encode homologues of cellular GPCRs. Human CMV (HCMV) carries four such genes: US27, US28, UL33 and UL78 (Chee et al., 1990a, b). Only two of these, UL33 and UL78, have been found to have counterparts in rodent CMVs: rat CMV (RCMV; R33 and R78, respectively; Beisser et al., 1998, 1999; Vink et al., 1999, 2001) and murine CMV (MCMV; M33 and M78, respectively; Davis-Poynter et al., 1997; Rawlinson et al., 1996).

The fact that the HCMV UL33 gene has homologues in all currently known betaherpesvirus genomes underlines the biological relevance of the UL33 gene family. The best-characterized members of this family are HCMV UL33 (Chee et al., 1990a, b; Waldhoer et al., 2002), MCMV M33 (Davis-Poynter et al., 1997; Rawlinson et al., 1996; Waldhoer et al., 2002), RCMV R33 (Beisser et al., 1998; Gruijthuijsen et al., 2002; Kaptein et al., 2003) and the U12 genes of human herpesvirus (HHV)-6A (Gompels et al., 1995), HHV-6B (Dominguez et al., 1999; Isegawa et al., 1998) and HHV-7 (Nakano et al., 2003; Nicholas, 1996). The biological significance of the UL33-like genes has previously been demonstrated in studies using recombinant CMVs that carry either a disrupted M33 (Davis-Poynter et al., 1997) or R33 gene (Beisser et al., 1998) in their genomes. In cell culture, each of these mutant viruses replicated with similar efficiency to the corresponding wild-type (WT) viruses (Beisser et al., 1998; Davis-Poynter et al., 1997; Margulies et al., 1996). However, during in vivo infection, significant differences were observed between animals infected with the recombinants and those infected with the WT viruses. In contrast to their WT counterparts, M33- and R33-deleted viruses could not be detected within the salivary glands of infected mice and rats, respectively, indicating that M33 and R33 play a role in virus dissemination to, or replication in, the salivary glands (Beisser et al., 1998; Davis-Poynter et al., 1997). Furthermore, it was shown that, in the RCMV/rat model, R33 also has a more general function: a significantly lower mortality was seen among rats infected with R33-deleted RCMV compared with those infected with WT RCMV (Beisser et al., 1998).

The predicted amino acid sequences of the proteins encoded by the UL33-like genes have been found to comprise several features characteristic of a distinct subfamily of GPCRs, the chemokine receptors (Beisser et al., 1998; Davis-Poynter et al., 1997). In accordance with these characteristics, both HHV-6B pU12 and HHV-7 pU12 have been reported to act as chemokine receptors. In response to binding of various CC chemokines, HHV-6B pU12 mediated the release of calcium from intracellular stores (Isegawa et al., 1998). Recently, Nakano et al. (2003) demonstrated that HHV-7 pU12 is also a calcium-mobilizing receptor in response to binding of CC chemokine macrophage inhibitory protein 3{beta}. In contrast to the pU12 proteins of HHV-6B and HHV-7, the other pUL33 family members have not been demonstrated to bind chemokines. Nevertheless, pUL33 and pR33 as well as pM33 are functional GPCRs, as they signal in a ligand-independent, constitutive fashion, activating a broad range of G proteins (Gruijthuijsen et al., 2002; Waldhoer et al., 2002).

Constitutive signalling by pR33 is marked by activation of G proteins of the Gq/11 and the Gi/0 class. The pR33-mediated activation of Gq/11 stimulates phospholipase C (PLC) resulting in an intracellular rise of diacylglycerol and inositol phosphates (InsPs). Constitutive activation of pertussis toxin (PTX)-sensitive Gi/0 proteins by pR33 inhibits adenylate cyclase and results in a reduced activation of cyclic AMP-responsive element (CRE)-driven transcription. At the same time, the interaction of pR33 with Gi/0 enhances NF-{kappa}B activation and co-stimulates PLC activity (Gruijthuijsen et al., 2002). Constitutive signalling by HCMV pUL33 differs in some respects from that of pR33. Both receptors stimulate PLC but, like pM33, pUL33 enhances CRE-mediated transcription via the activation of p38 mitogen-activated protein kinase, as opposed to the pR33-mediated inhibition of CRE-driven transcription (Gruijthuijsen et al., 2002; Waldhoer et al., 2002).

Currently, it is well established that the pUL33-like receptors signal in a constitutive fashion and are capable of influencing various signalling pathways in vitro. Nevertheless, data concerning structure–function relationships are lacking for these receptors. We therefore set out to identify structural determinants that are responsible for the differential constitutive activity of these proteins. To this purpose, a series of pR33 point and truncation mutants were generated and tested for cellular localization and constitutive activity in various signal transduction assays. Additionally, a panel of six pR33/pUL33 chimeric receptors was generated in which each of the intracellular loops was exchanged to create either ‘gain’ or ‘loss of function’ mutants with respect to constitutive signalling to the CRE.


   METHODS
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cell culture and transfection.
COS-7 cells (ATCC CRL-1651) were cultured and transfected as described previously (Casarosa et al., 2001).

DNA constructs.
Expression vectors pcDNA3/EGFP, pcDNA3/R33, pcDNA3/R33-EGFP, pcDNA3/UL33 and pcDNA/UL33-EGFP have been described previously (Casarosa et al., 2003; Gruijthuijsen et al., 2002). Reporter plasmid pTLNC-21CRE (Fluhmann et al., 1998) was obtained from Dr W. Born (National Jewish Medical and Research Centre, Denver, CO, USA). The pNF-{kappa}B-Luc vector was purchased from Stratagene.

Plasmids pcDNA3/N130D-EGFP, pcDNA3/N130A-EGFP, pcDNA3/R131A-EGFP, pcDNA3/Y132A-EGFP, pcDNA3/Y132F-EGFP, pcDNA3/R133A-EGFP, pcDNA3/R327A-EGFP and pcDNA3/R328A-EGFP encoding C-terminal EGFP-tagged pR33 point mutants were generated by primer-directed mutagenesis using plasmid pUC119/R33-EGFP as the template. Plasmid pUC119/R33-EGFP was constructed by digestion of plasmid pcDNA3/R33-EGFP with KpnI and XbaI, followed by subsequent cloning of the 2·3 kb fragment containing the R33EGFP open reading frame (ORF) into the corresponding sites of pUC119. Primer-directed mutagenesis was essentially carried out as described by Deng & Nickoloff (1992) using the ScaI/StuI primer and the N130D, N130A, R131A, Y132F, Y132A, R133A, R327A or R328A primer (Table 1). The resulting point-mutated pUC119/R33-EGFP derivatives were BamHI and NotI digested and each 2·3 kb fragment containing the respective point-mutated R33–EGFP ORF was cloned in the corresponding sites of pcDNA3.


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Table 1. PCR primers used in this study

 
Plasmids pcDNA3/N130D, pcDNA3/N130A, pcDNA3/R131A, pcDNA3/Y132A, pcDNA3/Y132F, pcDNA3/R133A, pcDNA3/R327A and pcDNA3/R328A, which encoded untagged pR33 point mutants, were generated by Eco47III and XbaI digestion of pcDNA3/N130D-EGFP, pcDNA3/N130A-EGFP, pcDNA3/R131A-EGFP, pcDNA3/Y132A-EGFP, pcDNA3/Y132F-EGFP, pcDNA3/R133A-EGFP, pcDNA3/R327A-EGFP or pcDNA3/R328A-EGFP, respectively, followed by Klenow treatment and self ligation of the linearized plasmids. In this way, the 0·8 kb Eco47III–XbaI fragment containing the EGFP sequence was removed and an in-frame stop codon was introduced at the 3' end of the mutated R33 ORF in each plasmid.

Expression vectors pcDNA3/C{Delta}13, pcDNA3/C{Delta}44 and pcDNA3/C{Delta}61 encoding pR33 mutants with C-terminal truncations of 13 (C{Delta}13), 44 (C{Delta}44) and 61 (C{Delta}61) aa, respectively, were generated by PCR with primers FR33X and RR33C{Delta}13, RR33C{Delta}44 or RR33C{Delta}61, respectively (Table 1), using pcDNA3/R33 as the template. Subsequently, expression vectors pcDNA3/C{Delta}13-EGFP, pcDNA3/C{Delta}44-EGFP and pcDNA3/C{Delta}61-EGFP, encoding the respective truncated receptors with a C-terminal EGFP tag, were constructed by NheI and XbaI digestion of plasmid p368 (Gruijthuijsen et al., 2002) and subsequent in-frame cloning of the 768 bp fragment containing the EGFP sequence, in the unique NheI site at the 3' end of the mutated R33 ORF in plasmids pcDNA3/C{Delta}13, pcDNA3/C{Delta}44 and pcDNA3/C{Delta}61, respectively.

Expression vectors pcDNA3/R33i1, pcDNA3/R33i2, pcDNA3/R33i3, pcDNA3/UL33i1, pcDNA3/UL33i2 and pcDNA3/UL33i3 encoded either pR33- or pUL33-based chimeric receptors, in which either the first, second or third intracellular loop was replaced by the corresponding loop of pUL33 or pR33, respectively (see Figs 1 and 5). The chimeric sequences were generated by means of an overlapping PCR procedure. For the pR33-based chimeras, this PCR procedure was carried out with primers FR33X, RR33E and overlapping primer pair RRi1/FRi1, RRi2/FRi2 or RRi3/FRi3 (Table 1), using pcDNA3/R33 and pcDNA3/UL33 as templates. For the pUL33-based chimeras, the PCR procedure was carried out with primers FUL33, RUL33E and overlapping primer pair RULi1/FULi1, RULi2/FULi2 or RULi3/FULi3 (Table 1), again using pcDNA3/R33 and pcDNA3/UL33 as templates.



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Fig. 1. Alignment of pR33 and pUL33 amino acid sequences. Black and grey boxes highlight identical and similar amino acid residues, respectively. Black dots above the sequences indicate pR33 residues that were been point mutated in this study. The residues that were swapped between pR33 and pUL33 to generate chimeric receptors pR33i1 and pUL33i1 (i1), pR33i2 and pUL33i2 (i2), pR33i3 and pUL33i3 (i3) are indicated by a black line below the sequences. The final C-terminal residue of each truncated pR33 mutant is indicated by an arrow and shows the number of residues that were deleted from the sequence.

 


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Fig. 5. Expression and activity of pR33/pUL33 chimeric receptors. (A–H) Expression of C-terminal EGFP-tagged pR33, pUL33 and pR33/pUL33 chimeras. COS-7 cells were transiently transfected with pcDNA3/R33-EGFP (A), pcDNA3/R33i-EGFP (B), pcDNA3/R33i2-EGFP (C), pcDNA3/R33i3-EGFP (D), pcDNA3/UL33-EGFP (E), pcDNA3/UL33i1-EGFP (F), pcDNA3/UL33i2-EGFP (G) or pcDNA3/UL33i3-EGFP (H) (2 µg per 106 cells), fixed after 48 h and subjected to confocal microscopy. A schematic representation of each chimeric amino acid sequence is given below the respective confocal pictures. The amino acid sequences of pUL33 and pR33 are depicted in black and white, respectively. (I–K) Characterization of constitutive signalling by pR33, pUL33 and pR33/pUL33 chimeras in the InsP accumulation assay (I), forskolin-stimulated, CRE-driven luciferase expression assay (J) and unstimulated CRE-driven luciferase expression assay (K). Cells were (co)transfected with pcDNA3 (control), pcDNA3/R33, pcDNA3/UL33, pcDNA3/R33i1, pcDNA3/R33i2, pcDNA3/R33i3, pcDNA3/UL33i1, pcDNA3/UL33i2 or pcDNA3/UL33i3 [2 µg (I, K) or 0·5 µg (J) per 106 cells]. The assays were performed in either the presence (+) or absence (-) of pertussis toxin (PTX).

 
Plasmids pcDNA3/R33i1-EGFP, pcDNA3/R33i2-EGFP, pcDNA3/R33i3-EGFP, pcDNA3/UL33i1-EGFP, pcDNA3/UL33i2-EGFP and pcDNA3/UL33i3-EGFP encoding C-terminal EGFP-tagged chimeric receptors were generated in a similar fashion, as described above for the truncation mutant expression constructs. The integrity of all DNA constructs was verified by sequence analysis.

Confocal imaging.
Transiently transfected cells (COS-7) were grown on glass coverslips. After 48 h, the cells were fixed for 10 min with 3·7 % formol in PBS and the coverslips were mounted for subsequent confocal imaging. Confocal images were collected at a wavelength of 488 nm and processed as described previously (Gruijthuijsen et al., 2002).

Signal transduction assays.
Inositol phosphate production in transfected COS-7 cells was determined as described previously (Gruijthuijsen et al., 2002). For the reporter gene assays, COS-7 cells were transiently transfected with either reporter plasmid pNF-{kappa}B-Luc (NF-{kappa}B assay) or pTLNC-21CRE (CRE assay) (5 µg per 106 cells) and one of the GPCR-expressing plasmids. Transfected cells were seeded in 96-well white plates (Costar) with serum-free medium in either the presence or absence of PTX (100 ng ml-1). Luciferase activity was measured 48 h after transfection for cells transfected with the NF-{kappa}B reporter construct. For cells transfected with the CRE reporter construct, luciferase activity was assayed 30 h after transfection, either with or without stimulation with forskolin at a concentration of 10 µM for 6 h, as described previously (Gruijthuijsen et al., 2002).

Statistical analysis.
All data shown are expressed as mean±SE. Statistical analysis was carried out using Student's t-test. Values of P<0·05 were considered to indicate a significant difference.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Constitutive signalling by pR33 C-terminal truncation mutants
In general, the intracellular C-tail of GPCRs is an important determinant of the signalling activity of these proteins. In the case of the pUL33 family members, the C-terminal sequences are highly divergent. Previously, we reported the C-tail of pR33 to comprise a unique motif consisting of 11 consecutive prolines (Beisser et al., 1998). To investigate the role of this proline stretch in pR33-mediated signalling, we generated truncated pR33 variants, C{Delta}13, C{Delta}44 and C{Delta}61, which lacked 13, 44 and 61 aa, respectively, from the C terminus (Fig. 1). To monitor receptor expression and localization, each truncated mutant was also tagged C-terminally with EGFP. Expression of these tagged mutants, as well as that of EGFP and pR33–EGFP, was studied by confocal microscopy of transiently transfected COS-7 cells (Fig. 2). Fluorescence within cells expressing EGFP was seen dispersed throughout the nucleus and cytoplasm (Fig. 2A). However, in cells expressing either pR33–EGFP (Fig. 2B), C{Delta}13–EGFP (Fig. 2C) or C{Delta}44–EGFP (Fig. 2D), fluorescence clearly co-localized with the cell membrane, as well as with intracellular perinuclear vesicles. This indicated that the mutant receptors C{Delta}13–EGFP and C{Delta}44–EGFP, like pR33-EGFP, were correctly expressed on the surface of transfected cells. In contrast, the fluorescent signal within cells expressing mutant receptor C{Delta}61–EGFP did not co-localize with the cell surface, but appeared to be retained intracellularly (Fig. 2E).



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Fig. 2. Constitutive signalling by pR33 C-terminal truncation mutants. (A–E) Expression of EGFP and C-terminal EGFP-tagged pR33 and pR33 mutants. COS-7 cells were transiently transfected with either pcDNA3/EGFP (A), pcDNA3/R33-EGFP (B), pcDNA3/C{Delta}13-EGFP (C), pcDNA3/C{Delta}44-EGFP (D) or pcDNA3/C{Delta}61-EGFP (E) (2 µg per 106 cells), fixed after 48 h and subjected to confocal microscopy. Below the confocal images, schematic representations of the truncated mutants are given. (F) Induction and accumulation of InsP. COS-7 cells (1x106) were transiently transfected with either pcDNA3, pcDNA3/R33 pcDNA3/C{Delta}13, pcDNA3/C{Delta}44 or pcDNA3/C{Delta}61 (2 µg per 106 cells) and InsP accumulation was measured 48 h after transfection. (G, H) NF-{kappa}B- and CRE-driven luciferase reporter gene assays. COS-7 cells (1x106) were transiently co-transfected with either pNF-{kappa}B-Luc (G) or pTLNC-21CRE (H) and with either pcDNA3 (control), pcDNA3/R33, pcDNA3/C{Delta}13, pcDNA3/C{Delta}44 or pcDNA3/C{Delta}61 [2 µg (G) or 0·5 µg (H) per 106 cells]. For the NF-{kappa}B assay, luciferase activity was measured 48 h after transfection. For the CRE assay, cells were stimulated with forskolin 24 h after transfection and luciferase activity was measured 30 h after transfection. Experiments were carried out in either the presence (+) or absence (-) of pertussis toxin (PTX).

 
Subsequently, the signalling characteristics of the truncated receptors C{Delta}13, C{Delta}44 and C{Delta}61 were compared with those of pR33 using the InsP accumulation assay (Fig. 2F) as well as the NF-{kappa}B- and CRE-driven reporter gene expression assays (Fig. 2G and H, respectively). As described previously (Gruijthuijsen et al., 2002), pR33 constitutively activated the production of InsP (Fig. 2F). Cells expressing pR33 showed an approximately twofold increase in InsP accumulation compared with basal InsP accumulation in cells transfected with an ‘empty’ vector (pcDNA3). A similar increase in InsP accumulation was seen for cells expressing the truncated receptors C{Delta}13 and C{Delta}44. Cells expressing C{Delta}61, however, showed a level of InsP accumulation similar to that of mock-transfected cells.

As mentioned above, pR33 constitutively enhances NF-{kappa}B-mediated transcription and reduces CRE-mediated transcription by activation of PTX-sensitive Gi/0 proteins. Fig. 2(G) shows that, within cells co-transfected with the NF-{kappa}B reporter gene construct and either pcDNA3/R33, pcDNA3/C{Delta}13 or pcDNA3/C{Delta}44, luciferase activity levels were four to five times higher than in cells co-transfected with an empty vector. As expected, the luciferase activity level within these cells was dramatically lower in the presence of PTX. In contrast, in cells co-transfected with the NF-{kappa}B reporter gene plasmid and pcDNA3/C{Delta}61, luciferase activity levels were similar to those in mock-transfected cells, in either the presence or the absence of PTX.

In the CRE reporter gene assay (Fig. 2H), the luciferase activity levels in cells expressing pR33, pC{Delta}13 or pC{Delta}44 were approximately 40 % of the levels in mock-transfected cells. The inhibitory effect of these proteins could be completely blocked by the addition of PTX. As in the other assays, mutant pC{Delta}61 did not show any activity in the CRE reporter gene assay. In all three assays, the EGFP-tagged receptors displayed similar activities to their native counterparts (data not shown).

Taken together, these data demonstrated that deletion of up to 44 aa from the C terminus of pR33 influences neither expression nor constitutive activity of the protein. This shows that the stretch of 11 consecutive proline residues at aa 375–385 is dispensable for pR33 signalling to PLC, NF-{kappa}B and CRE in vitro. Deletion of 61 aa from the C-terminus, however, resulted in an inactive mutant (C{Delta}61). This inactivity was most likely due to intracellular retention of the protein, as demonstrated for its EGFP-tagged counterpart, C{Delta}61–EGFP (Fig. 2E). Therefore, we hypothesize that the sequence between aa 327 and 344 contains residues critical for correct pR33 folding and/or cell-surface expression.

The pR33 C-terminal RR motif
Previously, we found that the C terminus of pUL33 could replace that of pR33 without affecting expression or signalling of pR33 in vitro (Casarosa et al., 2003). This finding is remarkable in light of the low level of sequence similarity between the C termini of pR33 and pUL33. The alignment of pR33 and pUL33 (Fig. 1) shows that the pR33 sequence between aa R327 and P344, which represents the C-terminal amino acids of C{Delta}61 and C{Delta}44, respectively, contains only five residues that are conserved between pR33 and pUL33. Two of these residues, R327 and R328, form a basic motif that is highly conserved among the pR33 family members (Vink et al., 2001). It has been shown that positively charged motifs in the C terminus of the chemokine receptor CCR5 (Venkatesan et al., 2001) play a role in correct receptor folding and cell-surface expression. Therefore, we investigated the function of these conserved arginines in pR33 expression and signalling by the generation of two point mutants, R327A and R328A. First, the expression of EGFP-tagged versions of these point mutants (R327A–EGFP and R328A–EGFP) was monitored by confocal microscopy. In the majority of either R327A–EGFP- or R328A–EGFP-expressing cells (Fig. 3A and D, respectively), fluorescence appeared to be confined to intracellular compartments, indicating that the mutant receptors were not correctly expressed on the cell surface. However, in a small proportion of transfected cells, corresponding to approximately 5 % for R327A–EGFP and 10 % for R328A–EGFP, fluorescence was found to co-localize with the cell membrane as well as with intracellular vesicles (Fig. 3B and C, respectively), as was observed for pR33–EGFP (Fig. 2B). This suggested that the mutant receptors were expressed on the cell surface in only a small proportion of the transfected cells. Interestingly, the inefficient cell-surface expression of R327A–EGFP and R328A–EGFP was reflected in the activity of these mutants in the signal transduction assays. Cells expressing R327A did not show a statistically significant increase in InsP accumulation levels compared with mock-transfected cells. However, R328A (or its EGFP-tagged version; data not shown) did display a slight but significant activity in this assay (Fig. 3E). This showed that R328A and R328A–EGFP were capable of constitutively activating PLC, despite their low level of cell-surface expression. Interestingly, both R327A and R328A showed activity in the NF-{kappa}B reporter gene assay. Although these mutants induced lower levels of NF-{kappa}B-mediated transcription than pR33, these levels were significantly higher than those in mock-transfected cells (Fig. 3F). Moreover, the activity of R327A and R328A in the NF-{kappa}B assay could be blocked by PTX, indicating that these mutants, like pR33, constitutively activate Gi/0 proteins. The activation of these G proteins was confirmed in the CRE assay, in which both R327A and R328A inhibited CRE-mediated transcription in a PTX-sensitive fashion (Fig. 3G). In all assays, similar signalling results were obtained for either the EGFP-tagged or untagged version of each mutant (data not shown). Taking these findings together, we conclude that the conserved, basic RR motif within the C terminus of pR33 is important for correct pR33 folding and/or cell-surface expression. We deem it unlikely, however, that this motif is involved in pR33-mediated constitutive signalling, since the small proportion of the R327A and R328A receptors that did appear to be expressed on the cell surface showed signalling to NF-{kappa}B and CRE similar to pR33.



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Fig. 3. Characterization of the pR33 RR motif. (A–D) Expression of mutants R327A and R328A. COS-7 cells were transiently transfected with either pcDNA3/R327A-EGFP (A, B) or pcDNA3/R328-EGFP (C, D) (2 µg per 106 cells), fixed after 48 h and subjected to confocal microscopy. Below the confocal images, schematic representations of the amino acid sequences of the mutants are given. The black dots indicate residues that were point mutated. (E–G) Constitutive signalling of the mutants in the InsP accumulation assay (E) and the NF-{kappa}B- (F) and CRE-driven (G) luciferase reporter gene assays. COS-7 cells were transiently (co)transfected with either pcDNA3 (control), pcDNA3/R33, pcDNA3/R327A or pcDNA3/R328 [2 µg (F, G) or 0·5 µg (H) per 106 cells] and analysed in the different assays. Experiments were carried out in either the presence (+) or absence (-) of pertussis toxin (PTX).

 
The pR33 NRYR motif
The highly conserved DRY motif, located within the second intracellular (i2) loop of GPCRs, generally determines the efficiency of G protein activation as well as the selectivity of receptor/G protein interactions (for a review, see Wess, 1998). Moreover, constitutive activity displayed by various (mutant) receptors has been linked to the presence of different residues at the aspartic acid position within this motif (Burger et al., 1999; Scheer et al., 1997; Wess, 1998). Interestingly, the predicted amino acid sequence of pR33 contains an NRY motif instead of a DRY motif (Fig. 1). Another interesting residue in the i2 loop of pR33 is the arginine that directly follows the NRY motif (R133; Fig. 1). Although a positively charged residue is found at the same position within all pR33 family members, it is uncommon in other GPCRs, which instead contain a hydrophobic residue at this position (http://www.gpcr.org/7tm).

To evaluate the role of the pR33 NRYR motif in constitutive activity and G-protein selectivity, the following single-point mutants were generated: N130D, N130A, R131A, Y132F, Y132A and R132A. To monitor expression, each mutant receptor was also tagged at its C terminus with EGFP. Confocal microscopy of transfected cells showed that the fluorescence patterns of all EGFP-tagged mutant receptors were similar to the fluorescence pattern of pR33-EGFP (compare Fig. 4A–F with Fig. 2B). This suggested that each mutant receptor was correctly expressed on the cell membrane.



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Fig. 4. Characterization of the pR33 NRYR motif. (A–F) Expression of C-terminal EGFP-tagged pR33 NRYR point mutants. COS-7 cells were transiently transfected with pcDNA3/N130D-EGFP (A), pcDNA3/N130A-EGFP (B), pcDNA3/R131A-EGFP (C), pcDNA3/Y132F-EGFP (D), pcDNA3/Y132A-EGFP (E) or pcDNA3/R133A-EGFP (F) (2 µg per 106 cells), fixed after 48 h and subjected to confocal microscopy. The schematic diagrams below the confocal pictures indicate the positions of the single-point mutations (black dots) in the respective amino acid sequences. (G–I) Constitutive signalling of the mutants in the InsP accumulation assay (G) and the NF-{kappa}B- (H) and CRE-driven (I) luciferase reporter gene assays. COS-7 cells were transiently (co)transfected with pcDNA3 (control), pcDNA3/R33, pcDNA3/N130D, pcDNA3/N130A, pcDNA3/R131A, pcDNA3/Y132F, pcDNA3/Y132A or pcDNA3/R133A [2 µg (G, H) or 0·5 µg (I) per 106 cells] and analysed in the different assays. Experiments were carried out in either the presence (+) or absence (-) of pertussis toxin (PTX).

 
Next, we compared the signalling characteristics of the mutant receptors with those of pR33 in the InsP assay, as well as in the NF-{kappa}B and CRE reporter gene assays. As can be seen in Fig. 4(G–I), receptors N130D, Y132F, Y132A and R133A displayed similar signalling profiles to pR33. Like pR33, each of these mutants induced both an increase in InsP production (Fig. 4G) and a PTX-sensitive increase in NF-{kappa}B-mediated transcription (Fig. 4H). In addition, these receptors mediated a PTX-sensitive decrease in CRE-mediated transcription (Fig. 4I). Despite the similarities between pR33 and mutants Y132F, Y132A and R133A in their signalling profiles, a lower level of activity was observed in cells expressing the mutants compared with cells expressing the WT protein. In contrast, mutant N130D induced levels of InsP production, as well as NF-{kappa}B- and CRE-mediated transcription, comparable with pR33 (Fig. 4G–I). This demonstrated that mutation of N130 to an aspartic acid residue, which is found in most GPCRs, does not alter the constitutive signalling characteristics of pR33. However, mutation of N130 to alanine resulted in a dramatic change in these characteristics: mutant N130A was found to be inactive in the InsP accumulation assay (Fig. 4G). This indicated that this mutant was impaired in its ability to activate PLC. Since the activation of PLC by pR33 is mediated mainly via coupling to Gq/11 proteins, it is likely that mutant N130A is unable to activate this class of G proteins. Surprisingly, mutant N130A did show activity in both the NF-{kappa}B and CRE reporter gene assays. Although the signalling activity of N130A in the NF-{kappa}B assay was lower than that of pR33 (Fig. 4H), the activity of this mutant in the CRE assay was similar to that of the WT receptor (Fig. 4I). Moreover, the activity of N130A in these assays could be blocked by the addition of PTX. This demonstrated that, although mutant receptor N130A appears unable to activate Gq/11 proteins, it is able to activate proteins of the Gi/0 class, resulting in activity in both the NF-{kappa}B and CRE reporter gene assays.

Only one of the NRYR point mutants, R131A, was found to be inactive in all signal transduction assays tested (Fig. 4G–I). Since R131A–EGFP displayed a similar expression pattern to pR33, we deem it unlikely that the inactivity observed for R131A and R131A–EGFP (data not shown) was due to intracellular retention. We therefore conclude that residue R131 is critical for constitutive signalling by pR33. Finally, the signalling characteristics of all EGFP-tagged mutants were comparable with those of their non-tagged counterparts (data not shown).

Expression and activity of pR33/UL33 chimeric receptors
As mentioned in the Introduction, pUL33 and pR33 show somewhat different signalling profiles (Gruijthuijsen et al., 2002; Waldhoer et al., 2002). A widely used approach to study differences in signalling activity between closely related receptors is to characterize chimeras of these proteins. We therefore constructed both pR33-based and pUL33-based chimeric receptors, in which either the first, second or third intracellular loop was exchanged (see Fig. 5B–D and F–H for a schematic representation of these chimeric receptors). To monitor receptor expression, we also generated C-terminal EGFP-tagged versions of the chimeras. As shown in Fig. 5(B and D), chimeric proteins pR33i1–EGFP and pR33i3–EGFP displayed a similar pattern of expression to pR33–EGFP (Fig. 5A). However, the expression of pR33i3–EGFP near the cell membrane was not as pronounced as that of either pR33–EGFP or pR33i1–EGFP. The third pR33-based chimera that we generated, pR33i2–EGFP, did not show localization at the cell surface. Instead, this protein was found dispersed throughout the cytoplasm of the transfected cells (Fig. 5C). Fluorescence in cells expressing either pUL33i2–EGFP (Fig. 5G) or pUL33–EGFP (Fig. 5E) co-localized with the cell membrane as well as with intracellular compartments. Fluorescence of pUL33i2–EGFP, however, was less pronounced at the cell surface and more intense in intracellular compartments compared with the fluorescence of pUL33–EGFP. In contrast, fluorescence within cells expressing either pUL33i1–EGFP (Fig. 5F) or pUL33i3–EGFP (Fig. 5H) did not co-localize with the cell membrane, but was confined to the nucleus and other intracellular compartments, indicating that these proteins are not correctly expressed on the cell surface.

Since pR33 and pUL33 both stimulate InsP production by constitutive activation of PLC, the functionality of the pR33/pUL33 chimeric receptors was first evaluated in the InsP accumulation assay. Fig. 5(I) shows that pR33-based chimera pR33i1 had a similar activity to pR33 in the InsP accumulation assay. In contrast, chimera pR33i3 displayed a much lower level of InsP production than pR33. Of the pUL33-based chimeric receptors, only pUL33i2 was found to be active in the InsP accumulation assay, although the level of activity of this receptor was significantly lower than that of pUL33 (Fig. 5I). As expected, the chimeras in which the EGFP-tagged variants did not show appropriate cell-surface expression, i.e. pR33i2, pUL33i1 and pUL33i3, were inactive in the InsP assay. Again, the EGFP-tagged receptors showed similar activities to their untagged counterparts (data not shown).

Next, we evaluated the activity of the chimeric receptors in the CRE assay. Like native pR33, chimeras pR33i1 and pR33i3 both induced a PTX-sensitive inhibition of CRE-mediated transcription (Fig. 5J), indicating that both chimeras activate Gi/0 proteins. However, as in the InsP accumulation assay, chimera pR33i2 was inactive in the CRE assay (data not shown). As shown previously (Waldhoer et al., 2002), pUL33 induced an increase in CRE-mediated transcription (Fig. 5K). Moreover, by eliminating the activation of Gi/0 proteins through the addition of PTX, the CRE-driven transcription levels within cells expressing pUL33 are stimulated even further (Casarosa et al., 2003). Although chimera pUL33i2 only induced a slight increase in the level of CRE-mediated transcription, this level was further increased in the presence of PTX, as observed for pUL33 (Fig. 5K). This indicates that, like pUL33, pUL33i2 is capable of stimulating both CRE-inhibiting and CRE-activating pathways, resulting in an overall activating effect. As in the InsP assay, chimeric proteins pUL33i1 and pUL33i3 were found to be inactive in the CRE assay (data not shown). Since chimeras pR33i2–EGFP, pUL33i1–EGFP and pUL33i3–EGFP did not show cell-surface expression or activity in the signal transduction assays, it is likely that the inactivity of their untagged counterparts, pR33i2, pUL33i1 and pUL33i3, respectively, is due to intracellular retention.

The results generated with the chimeras are summarized in Table 2. Taken together, we found that in only three of the six cases, the exchange of intracellular loops between pR33 and pUL33 resulted in receptors that were properly expressed on the cell surface and, concomitantly, displayed constitutive signalling activity. Both intracellular regions 1 and 3 of pUL33 were demonstrated functionally to replace the corresponding regions in pR33 without affecting the signalling profile of pR33. The reciprocal exchange of i1 and i3 between pR33 and pUL33, however, resulted in receptors that were retained intracellularly. Only a single pUL33-based chimera, pUL33i2, displayed signalling activity. Although the activity of this mutant was relatively low, its signalling profile was similar to that of WT pUL33. Our data indicate that the three intracellular regions of pR33 and pUL33 do not independently determine the differential signalling profiles of pR33 and pUL33.


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Table 2. Expression and constitutive activity of pR33, pUL33 and the mutant receptors

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
RCMV pR33, MCMV pM33 and HCMV pUL33 are functional GPCRs that signal in a ligand-independent, constitutive fashion (Gruijthuijsen et al., 2002; Waldhoer et al., 2002). By activation of various classes of G proteins, these receptors modulate PLC activity as well as NF-{kappa}B- and CRE-mediated transcription in vitro. Previously, we reported that pR33 enhances PLC activity via stimulation of G proteins of the Gq/11 class. In addition, pR33 activates G proteins of the Gi/0 class, which results in an upregulation of NF-{kappa}B- and downregulation of CRE-mediated transcription (Gruijthuijsen et al., 2002). By mutational analysis of the pR33 NRY motif, we have identified two residues, N130 and R131, that are critical for constitutive G protein activation. Although replacement of N130 by an aspartic acid residue did not change the constitutive behaviour of the resulting mutant N130D, replacement of the residue by alanine did. In contrast to pR33, mutant N130A was unable to stimulate PLC. As pR33 predominantly stimulates PLC via activation of Gq/11 proteins, we concluded that N130A is impaired in its ability to activate Gq/11. In contrast, PTX-sensitive signalling of N130A to CRE was unaltered, indicating that the ability to activate Gi/0 was preserved. These data imply that N130 of pR33 is critical for activation of Gq/11 but not Gi/0. However, it appears that proper Gq/11 activation is not determined by the specific presence of an asparagine residue at position 130, but rather by the presence of a polar residue at this position, since mutant N130D was not impaired in PLC activation. It is known that the polar residues within DRY motifs are generally involved in stabilizing receptor conformations that form receptor/G protein interfaces (Wess, 1998). This stabilization is mediated by the formation of hydrogen bonds between polar residues of the DRY motif and polar residues in the other intracellular loops as well as transmembrane regions (Scheer et al., 1997). Therefore, substitution of the polar asparagine in pR33 by another polar residue, i.e. aspartic acid, might result in formation of similar hydrogen bonds to native pR33, thereby preserving the active conformation of the receptor. In contrast, replacement of the polar N130 residue by a non-polar residue, such as alanine, would be expected to alter the hydrogen bond network between the intracellular regions of pR33, which may have a profound effect on the receptor's conformation and, consequently, on its interaction with specific subtypes of G protein. Also, since the NRY motif is situated near the ‘bottom’ of the third transmembrane domain of pR33, the introduction of a hydrophobic residue at position N130 might pull the NRY motif further into the cellular membrane, making it less accessible for certain G proteins. Both mechanisms would predict an altered conformation of N130A compared with native pR33 or mutant N130D. While this altered conformation of N130A would result in a failure to activate Gq/11 proteins, it would still allow proper Gi/0 activation.

Previously, various class I GPCRs, including the chemokine receptor CXCR2, have been shown to gain constitutive activity upon substitution of the aspartic acid of the DRY motif by hydrophobic or neutral residues (Burger et al., 1999; Scheer et al., 1997; Wess, 1998). Interestingly, while a neutral residue is present at the aspartic acid position in the pR33 NRY motif, N130, this residue is not essential for constitutive signalling activity, as demonstrated by the WT characteristics of signalling by mutant N130D. Similar findings have been reported for the HHV-8 (Kaposi's sarcoma-associated herpesvirus) GPCR in which mutation of the corresponding hydrophobic residue valine (V142) to aspartic acid did not eliminate its constitutive activity (Rosenkilde et al., 2000). In addition, the results with our mutant N130D demonstrated that the asparagine residue of pR33 is not the structural determinant for the observed differences in constitutive signalling by pR33 and pUL33, as was previously suggested by Waldhoer et al. (2002). Instead, it is likely that residues other than N130 of pR33 (and D128 of pUL33) are responsible for the signalling differences between these receptors.

One of the residues from the pR33 NRY motif, R131, was found to be essential for constitutive activity of the receptor. A mutant in which R131 was replaced by alanine (R131A) was unable to stimulate either Gq/11- or Gi/0-mediated pathways. This observation is in agreement with the finding that the arginine from the DRY motif, as found in most class I GPCRs, is essential for high-affinity binding of G proteins (Wess, 1998).

Mutation of the tyrosine residue of the pR33 NRY motif to either phenylalanine or alanine did not result in receptors with characteristics significantly different from those of the WT protein. For some chemokine receptors, such as CCR2, the tyrosine residue was found to be crucial for ligand-induced Gi/0-mediated responses (Damaj et al., 1996; Mellado et al., 1998; Rodriguez-Frade et al., 1999). Currently, it is not possible to test whether or not pR33 residue Y132 plays a similar role, since a ligand for pR33 has not been identified. Nevertheless, we may conclude that the tyrosine residue of the NRY motif is not essential for constitutive activation of Gi/0 and Gq/11 by pR33.

Within the pR33 amino acid sequence, a basic residue, R133, follows the NRY motif. A basic residue is also present at the corresponding positions within the betaherpesvirus homologues of pR33 (Vink et al., 2001). Interestingly, most other GPCRs contain a hydrophobic residue at the position corresponding to R133 (http://www.gpcr.org/7tm). It has been reported that the hydrophobic residue following the DRY motif might be involved in Gi/0 activation, as was demonstrated for CXCR1 (Damaj et al., 1996). Nevertheless, we found mutant R133A to have a similar profile of expression, as well signalling, to pR33.

By analysis of C-terminally truncated mutants of pR33, we were able to show that the C-terminal 44 aa of pR33, including the polyproline motif, are dispensable for constitutive signalling by pR33 in vitro. This supports the general idea that membrane-distal parts of the GPCR C-tail are not directly involved in efficient G-protein coupling (Wess, 1998). Interestingly, truncation of the pR33 C-tail, up to residue R327, resulted in a mutant that was not properly expressed on the cell surface, but was mainly retained intracellularly. This inefficient cell-surface expression could largely be attributed to the lack of residues R327 and R328. Point mutation of either of these residues resulted in mutants (R327A and R328A) that were predominantly retained intracellularly. Interestingly, R327 and R328 are conserved among the divergent C-terminal sequences of all pR33 family members. These residues form part of a larger conserved motif, RxxxxCxxGxLxxRRxxL, which is located between residues 315 and 331 of the pR33 amino acid sequence (Vink et al., 1999). Previously, the importance of basic residues within the C termini of GPCRs for correct cell-membrane expression has also been demonstrated for human chemokine receptors CCR5 and CCR2B (Venkatesan et al., 2001). It is possible that these positively charged basic residues are involved in the interaction of GPCRs with the negatively charged phospholipid heads in the cellular membrane. This interaction may subsequently allow palmitoylated cysteine residues to stabilize the GPCR/membrane association by anchoring into the membrane and facilitating correct receptor folding and transport (Venkatesan et al., 2001). Alternatively, the basic amino acid motifs may play a role in the interaction between the GPCRs and proteins that guide correct GPCR expression (reviewed by Brady & Limbird, 2002).

In contrast to pR33 (Gruijthuijsen et al., 2002), pUL33 was found to stimulate CRE-mediated transcription (Waldhoer et al., 2002). This activity of pUL33 could not be transferred to pR33 by replacing either the first or third intracellular loop of pR33 with those of pUL33. The chimeric receptors pR33i1 and pR33i3 displayed similar activities to native pR33. Additionally, we previously found that the stimulatory effect of pUL33 in the CRE assay was not solely determined by the C terminus (Casarosa et al., 2003). Therefore, we conclude that stimulation of CRE by pUL33 is mediated either by the second intracellular loop or by the interaction of multiple intracellular domains. A study on chimeric human endothelin receptors has suggested that the second intracellular loop is an absolute requirement for stimulation of cyclic AMP formation, whereas the third intracellular loop plays an ancillary role in co-stimulating cyclic AMP production (Takagi et al., 1995). However, we found that the second intracellular loop of pUL33 could be substituted by the corresponding loop of pR33, without eliminating the potential of pUL33 to stimulate CRE-mediated transcription. Although only a very slight basal stimulation of CRE-mediated transcription was observed for pUL33i2, a clear stimulatory effect was seen in the presence of PTX. This indicated that both CRE-inhibitory as well as CRE-stimulatory signal transduction pathways were constitutively activated. It is likely that the relatively low signalling activity of pUL33i2 towards CRE is due to a defect of this mutant in cell-surface expression. Although cell-surface receptor densities were not quantified in this study, it was apparent from confocal microscopy that the EGFP-tagged variant, pUL33i2–EGFP, displayed low levels of fluorescence co-localizing with the cell membrane. In agreement with this, mutant pUL33i2 showed a considerably lower level of PLC activation than pUL33. We conclude that activation of CRE by pUL33 is the consequence of the concerted action of multiple intracellular regions and possibly also transmembrane domains. To specify which domains are involved, future experiments will be directed at the generation of a broader panel of pR33/pUL33 chimeric receptors in which multiple intracellular domains as well as transmembrane regions will be exchanged. Three of the chimeric receptors we studied, pUL33i1, pUL33i3 and pR33i2, were not correctly expressed on the cell surface. Possibly, incorrect folding and/or transport of these chimeras caused this defect, which could be the consequence of the inability to form specific interactions between various domains within these proteins. Indeed, it is known that certain residues and domains within the intracellular loops of GPCRs interact to stabilize receptor conformation and ensure correct receptor folding (Wess, 1998). It was noted in our study that, in particular, those chimeric receptors that comprised the second intracellular loop of pUL33 in combination with either the first or the third loop of pR33 were intracellularly retained. Together, these observations suggest that the interactions between the intracellular loops within both pR33 and pUL33 are of importance for correct receptor folding and/or transport, but that the specific amino acid residues that are involved in these interactions are not conserved between pR33 and pUL33.

To date, it is clear that most herpesvirus-encoded GPCRs are capable of constitutively modulating a wide variety of intracellular signal transduction pathways (Casarosa et al., 2001; Geras-Raaka et al., 1998; Gruijthuijsen et al., 2002; Schwarz & Murphy, 2001; Waldhoer et al., 2002). However, despite extensive speculation, it is still unclear if and how these viruses benefit from these and possibly other activities mediated by their GPCRs. Here, we have demonstrated the complexity of the intramolecular interactions within pUL33-like GPCRs that are required for efficient signalling and cellular expression. Of particular interest is the finding that mutation of a single residue may result in impaired constitutive activity in specific intracellular signalling pathways. For example, alteration of residue N130 to alanine eliminated the ability of pR33 to constitutively activate Gq/11- but not Gi/0-mediated pathways. Introduction of such mutations in the RCMV genome may contribute to the identification of viral GPCR-mediated signalling routes that are crucial in the pathogenesis of virus infection.


   ACKNOWLEDGEMENTS
 
Y. K. G. is supported by a grant (no. 901-02-224) from the Netherlands Organization for Scientific Research (NWO, medical sciences). C. V. and M. J. S. are supported by fellowships from the Royal Netherlands Academy of Arts and Sciences (KNAW).


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Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 10 October 2003; accepted 17 December 2003.