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
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ABSTRACT |
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INTRODUCTION |
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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
. 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-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 structurefunction 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.
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METHODS |
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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-
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 R33EGFP ORF was cloned in the corresponding sites of pcDNA3.
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Expression vectors pcDNA3/C13, pcDNA3/C
44 and pcDNA3/C
61 encoding pR33 mutants with C-terminal truncations of 13 (C
13), 44 (C
44) and 61 (C
61) aa, respectively, were generated by PCR with primers FR33X and RR33C
13, RR33C
44 or RR33C
61, respectively (Table 1
), using pcDNA3/R33 as the template. Subsequently, expression vectors pcDNA3/C
13-EGFP, pcDNA3/C
44-EGFP and pcDNA3/C
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
13, pcDNA3/C
44 and pcDNA3/C
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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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-
B-Luc (NF-
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-
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.
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RESULTS |
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As mentioned above, pR33 constitutively enhances NF-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-
B reporter gene construct and either pcDNA3/R33, pcDNA3/C
13 or pcDNA3/C
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-
B reporter gene plasmid and pcDNA3/C
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
13 or pC
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
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 375385 is dispensable for pR33 signalling to PLC, NF-B and CRE in vitro. Deletion of 61 aa from the C-terminus, however, resulted in an inactive mutant (C
61). This inactivity was most likely due to intracellular retention of the protein, as demonstrated for its EGFP-tagged counterpart, C
61EGFP (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
61 and C
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 (R327AEGFP and R328AEGFP) was monitored by confocal microscopy. In the majority of either R327AEGFP- or R328AEGFP-expressing cells (Fig. 3
A 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 R327AEGFP and 10 % for R328AEGFP, 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 pR33EGFP (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 R327AEGFP and R328AEGFP 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 R328AEGFP were capable of constitutively activating PLC, despite their low level of cell-surface expression. Interestingly, both R327A and R328A showed activity in the NF-
B reporter gene assay. Although these mutants induced lower levels of NF-
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-
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-
B and CRE similar to pR33.
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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. 4AF with Fig. 2B
). This suggested that each mutant receptor was correctly expressed on the cell membrane.
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Only one of the NRYR point mutants, R131A, was found to be inactive in all signal transduction assays tested (Fig. 4GI). Since R131AEGFP displayed a similar expression pattern to pR33, we deem it unlikely that the inactivity observed for R131A and R131AEGFP (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. 5
BD and FH 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 pR33i1EGFP and pR33i3EGFP displayed a similar pattern of expression to pR33EGFP (Fig. 5A
). However, the expression of pR33i3EGFP near the cell membrane was not as pronounced as that of either pR33EGFP or pR33i1EGFP. The third pR33-based chimera that we generated, pR33i2EGFP, 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 pUL33i2EGFP (Fig. 5G
) or pUL33EGFP (Fig. 5E
) co-localized with the cell membrane as well as with intracellular compartments. Fluorescence of pUL33i2EGFP, however, was less pronounced at the cell surface and more intense in intracellular compartments compared with the fluorescence of pUL33EGFP. In contrast, fluorescence within cells expressing either pUL33i1EGFP (Fig. 5F
) or pUL33i3EGFP (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 pR33i2EGFP, pUL33i1EGFP and pUL33i3EGFP 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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DISCUSSION |
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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, pUL33i2EGFP, 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.
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ACKNOWLEDGEMENTS |
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Received 10 October 2003;
accepted 17 December 2003.