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Constitutive Signaling by Kaposi's Sarcoma-associated Herpesvirus G-protein-coupled Receptor Desensitizes Calcium Mobilization by Other Receptors

2001; Elsevier BV; Volume: 276; Issue: 10 Linguagem: Inglês

10.1074/jbc.m006359200

1083-351X

Monica Lupu‐Meiri, Randi B. Silver, Arnold Simons, Marvin C. Gershengorn, Yoram Oron,

Herpesvirus Infections and Treatments

We coexpressed Kaposi's sarcoma-associated herpesvirus G protein-coupled receptors (KSHV-GPCRs) with thyrotropin-releasing hormone (TRH) receptors or m1-muscarinic-cholinergic receptors in Xenopus oocytes and in mammalian cells. In oocytes, KSHV-GPCR expression resulted in pronounced (81%) inhibition (heterologous desensitization) of Ca2+-activated chloride current responses to TRH and acetylcholine. Similar inhibitions of cytoplasmic free Ca2+responses to TRH were observed in human embryonic kidney HEK 293 EM cells and in mouse pituitary AtT20 cells. Further study of oocytes showed that this inhibition was partially reversed by interferon-γ-inducible protein 10 (IP-10), an inverse agonist of KSHV-GPCR. The basal rate of 45Ca2+ efflux in oocytes expressing KSHV-GPCRs was 4.4 times greater than in control oocytes, and IP-10 rapidly inhibited increased45Ca2+ efflux. In the absence of IP-10, growth-related oncogene α caused a further 2-fold increase in45Ca2+ efflux. In KSHV-GPCR-expressing oocytes, responses to microinjected inositol 1,4,5-trisphosphate were inhibited by 74%, and this effect was partially reversed by interferon-γ-inducible protein 10. Treatment with thapsigargin suggested that the pool of calcium available for mobilization by TRH was decreased in oocytes coexpressing KSHV-GPCRs. These results suggest that constitutive signaling by KSHV-GPCR causes heterologous desensitization of responses mediated by other receptors, which signal via the phosphoinositide/calcium pathway, which is caused by depletion of intracellular calcium pools. We coexpressed Kaposi's sarcoma-associated herpesvirus G protein-coupled receptors (KSHV-GPCRs) with thyrotropin-releasing hormone (TRH) receptors or m1-muscarinic-cholinergic receptors in Xenopus oocytes and in mammalian cells. In oocytes, KSHV-GPCR expression resulted in pronounced (81%) inhibition (heterologous desensitization) of Ca2+-activated chloride current responses to TRH and acetylcholine. Similar inhibitions of cytoplasmic free Ca2+responses to TRH were observed in human embryonic kidney HEK 293 EM cells and in mouse pituitary AtT20 cells. Further study of oocytes showed that this inhibition was partially reversed by interferon-γ-inducible protein 10 (IP-10), an inverse agonist of KSHV-GPCR. The basal rate of 45Ca2+ efflux in oocytes expressing KSHV-GPCRs was 4.4 times greater than in control oocytes, and IP-10 rapidly inhibited increased45Ca2+ efflux. In the absence of IP-10, growth-related oncogene α caused a further 2-fold increase in45Ca2+ efflux. In KSHV-GPCR-expressing oocytes, responses to microinjected inositol 1,4,5-trisphosphate were inhibited by 74%, and this effect was partially reversed by interferon-γ-inducible protein 10. Treatment with thapsigargin suggested that the pool of calcium available for mobilization by TRH was decreased in oocytes coexpressing KSHV-GPCRs. These results suggest that constitutive signaling by KSHV-GPCR causes heterologous desensitization of responses mediated by other receptors, which signal via the phosphoinositide/calcium pathway, which is caused by depletion of intracellular calcium pools. Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor interferon-γ-inducible protein 10 thyrotropin-releasing hormone TRH receptor m1-muscarinic-cholinergic receptor inositol 1,4,5-trisphosphate green fluorescent protein growth-related oncogene α gastrin-releasing peptide Hanks' balanced salt solution KSHV-GPCR,1 the gene product of ORF74 in the KSHV (human herpesvirus 8) genome, is a GPCR homologous to human chemokine CXC receptor 2 (1Cesarman E. Nador R.G. Bai F. Bohenzky R.A. Russo J.J. Moore P.S. Chang Y. Knowles D.M. J. Virol. 1996; 70: 8218-8223Crossref PubMed Google Scholar, 2Arvanitakis L. Geras-Raaka E. Varma A. Gershengorn M.C. Cesarman E. Nature. 1997; 385: 347-350Crossref PubMed Scopus (577) Google Scholar). Our group demonstrated that rodent fibroblasts expressing KSHV-GPCRs formed tumors in mice (3Bais C. Santomasso B. Coso O. Arvanitakis L. Geras-Raaka E. Gutkind J.S. Asch A.S. Cesarman E. Gershengorn M.C. Mesri E.A. Nature. 1998; 391: 86-89Crossref PubMed Scopus (753) Google Scholar). Recently, transgenic mice in which the KSHV-GPCR gene was regulated by the human CD2 enhancer/promoter and locus control region were shown to develop angioproliferative lesions that resembled the lesions of KS (4Yang T.-Y. Chen S.-C. Leach M.W. Manfra D. Homey B. Wiekowski M. Sullivan L. Jenh C.-H. Narula S.K. Chensue S.W. Lira S.A. J. Exp. Med. 2000; 191: 445-453Crossref PubMed Scopus (361) Google Scholar). These reports make the mechanism of action of KSHV-GPCR interesting in terms of tumorigenesis. We reported that KSHV-GPCR exhibited marked constitutive, agonist-independent activity in mammalian cells (2Arvanitakis L. Geras-Raaka E. Varma A. Gershengorn M.C. Cesarman E. Nature. 1997; 385: 347-350Crossref PubMed Scopus (577) Google Scholar). We also demonstrated that the constitutive activity of the viral GPCR could be inhibited by IP-10, acting as an inverse agonist (5Geras-Raaka E. Varma A. Ho H. Clark-Lewis I. Gershengorn M.C. J. Exp. Med. 1998; 188: 405-408Crossref PubMed Scopus (127) Google Scholar). These results were later confirmed by Rosenkildeet al. (6Rosenkilde M.M. Kledal T.N. Bräuner-Osborne H. Schwartz T.W. J. Biol. Chem. 1999; 274: 956-961Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Last, we showed that signaling by KSHV-GPCR could be inhibited by GPCR-specific protein kinases and suggested that this may represent a form of homologous desensitization of this viral receptor (7Geras-Raaka E. Arvanitakis L. Bais C. Cesarman E. Mesri E.A. Gershengorn M.C. J. Exp. Med. 1998; 187: 801-806Crossref PubMed Scopus (70) Google Scholar). However, the effect of the constitutive activity of KSHV-GPCR on signaling by other receptors (heterologous desensitization) has not been previously investigated. To investigate the effect of constitutive signaling by KSHV-GPCR on other receptors that utilize the phosphoinositide/calcium signaling pathway, we studied the behavior of this receptor in Xenopusoocytes and in mammalian cells. Our results show that KSHV-GPCR causes marked desensitization of responses mediated by TRH-Rs and m1-Rs by inhibiting InsP3-stimulated mobilization of Ca2+. Defolliculated oocytes were obtained from mature Xenopus females, essentially as previously described (8Shapira H. Lupu-Meiri M. Gershengorn M.C. Oron Y. Biophys. J. 1990; 57: 1281-1285Abstract Full Text PDF PubMed Scopus (22) Google Scholar). In vitro transcribed cRNAs for wild type TRH-Rs, m1-Rs (1 ng/oocyte), gastrin-releasing peptide (GRP) receptors (0.23 ng/oocyte), and KSHV-GPCRs (1–3 ng/oocyte) were injected 48 h before the assay as described previously (9Oron Y. Gillo B. Straub R.E. Gershengorn M.C. Mol. Endocrinol. 1987; 1: 918-925Crossref PubMed Scopus (37) Google Scholar, 10Lupu-Meiri M. Biet-Or A. Christensen S.B. Oron Y. Cell Calcium. 1993; 14: 101-110Crossref PubMed Scopus (45) Google Scholar, 11Matus-Leibovitch N. Nussenzveig D.R. Gershengorn M.C. Oron Y. J. Biol. Chem. 1995; 270: 1041-1047Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Two-electrode voltage clamp measurements were performed at V H = −80 mV, as previously described (10Lupu-Meiri M. Biet-Or A. Christensen S.B. Oron Y. Cell Calcium. 1993; 14: 101-110Crossref PubMed Scopus (45) Google Scholar, 12Lupu-Meiri M. Shapira H. Matus-Leibovitch N. Oron Y. Pflugers Arch. 1990; 417: 391-397Crossref PubMed Scopus (18) Google Scholar). Chloride currents were continuously recorded. Intracellular injections of InsP3 were performed using a Drummond microinjector (volume 4.6–9.2 nl/oocyte) as previously described (13Shapira H. Lupu-Meiri M. Lipinsky D. Oron Y. Cell Calcium. 1996; 19: 201-210Crossref PubMed Scopus (8) Google Scholar). 45Ca2+ efflux studies were performed essentially as described previously (10Lupu-Meiri M. Biet-Or A. Christensen S.B. Oron Y. Cell Calcium. 1993; 14: 101-110Crossref PubMed Scopus (45) Google Scholar), with the following modifications. Oocytes were either loaded with45Ca2+ by overnight incubation in a small volume of ND96 (CaCl2 concentration reduced to 0.6 mm) in the presence of 2 mCi/ml of45CaCl2 or injected with ∼120,000 cpm of carrier-free 45CaCl2. At various times after injection, oocytes were rinsed in ND96 and incubated each in 10 μl of ND96. At the desired time intervals, a sample of 5 μl was withdrawn and replaced with 5 μl of ND96 containing the desired additions, whenever required. At the end of the experiment, the residual radioactivity in each oocyte was determined. All results were presented as fractional efflux rates (percentage of residual label/min). Calcium mobilization was assayed in HEK 293 EM cells and AtT20 pituitary cells by ratiometric determination of [Ca2+]i using fura-2 as previously described (14Grimberg H. Zaltsman I. Lupu-Meiri M. Gershengorn M.C. Oron Y. Br. J. Pharmacol. 1999; 126: 1097-1106Crossref PubMed Scopus (11) Google Scholar, 15Silver R.B. Methods Cell Biol. 1998; 56: 237-251Crossref PubMed Google Scholar). HEK 293 EM cells were transiently transfected with TRH receptor cDNA (0.25–1 μg/ml) or cotransfected with TRH-R (0.25–1 μg/ml) and KSHV-GPCR (1 μg/ml) cDNAs using the calcium phosphate method as described (16O'Dowd B.F. Lee D.K. Huang W. Nguyen T. Cheng R. Liu Y. Wang B. Gershengorn M.C. George S.R. Mol. Endocrinol. 2000; 14: 183-193Crossref PubMed Scopus (95) Google Scholar). After 48 h, cells were used in binding and in calcium mobilization experiments. [3H]MeTRH binding was performed in 24-well dishes as described (17Wang W. Gershengorn M.C. Endocrinology. 1999; 140: 4916-4919Crossref PubMed Scopus (34) Google Scholar). Cells expressing similar numbers of TRH-Rs were studied. Cells expressing TRH-Rs and KSHV-GPCRs expressed TRH-Rs at levels that were 103 ± 8.8% those in cells expressing TRH-Rs alone. For calcium measurements, cells grown to confluence (2 days after plating) on 22-mm square standard glass coverslips (number 1) and maintained in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum at 37 °C, 5% CO2 were loaded with the membrane-permeant form of the calcium indicator fura-2 acetoxymethyl ester (1 μm) for 20–30 min at 37 °C. After loading with the dye, cells were rinsed with HBSS, and the coverslip with the fura-2-loaded cells was attached to the bottom of a chamber and mounted on the stage of an inverted epifluorescence microscope (Nikon Diaphot). The cells in the chamber were bathed in HBSS (200 μl) and maintained at room temperature. Cells were visualized under transmitted light with a Nikon CF Fluor oil immersion objective (40×/1.3 NA) before starting the fluorescence measurements. After monitoring the basal 340/380 fluorescence signal in the cells in the field of view, TRH was added in a 100-μl volume to the 200-μl chamber volume to yield a final concentration of ∼1.0 μm. In certain experiments, calibration of the emitted fura-2 fluorescence signal from each cell in the field was performed in the presence of the Ca2+ionophore, ionomycin (10 μm), and either HBSS or HBSS with 10 mm EGTA. Intracellular Ca2+ levels were then calculated as described by Grynkiewicz et al. (18Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Each cell in the field of view was calibrated with its own corresponding R min andR max as previously described (15Silver R.B. Methods Cell Biol. 1998; 56: 237-251Crossref PubMed Google Scholar). Individual vials (50 μg) of the acetoxymethyl derivative of fura-2 were stored dry at 0 °C and reconstituted in dimethyl sulfoxide, at a concentration of 1 mm, for each experiment. The basic components of the experimental apparatus have been described previously (15Silver R.B. Methods Cell Biol. 1998; 56: 237-251Crossref PubMed Google Scholar, 19Cardone M.H. Smith B.L. Mennitt P.A. Mochly-Rosen D. Silver R.B. Mostov K.E. J. Cell Biol. 1996; 133: 997-1005Crossref PubMed Scopus (58) Google Scholar). Briefly, the imaging work station was controlled with the Metafluor software package (Universal Imaging, Westchester, PA). Image pairs were obtained every 0.1 s for the duration of the experiment at 340- and 380-nm excitation with emission at 510 nm. The fluorescence excitation was shuttered off except during the brief periods required to record an image. To check for interference from intrinsic autofluorescence and background, images were obtained on unloaded cells using the same exposure time and filter combinations used for the experiments and found to be an insignificant component of the fluorescence signal. To determine the effect of KSHV-GPCR expression on TRH responses in another mammalian cell line, AtT20 cells stably expressing TRH-Rs were transiently cotransfected with two vectors: pcDNA3.1(+), with KSHV-GPCR coding DNA inserted betweenXbaI and EcoRI restriction sites and pIT (20Simons, A. H, Dafni, N., Dotan, I., Oron, Y., and Canaani, D. (2001)Genome. Res. , 11, in press.Google Scholar) coding for GFP mutant Topaz®. The Topaz® GFP mutant has excitation-emission (514/527 nm) characteristics that do not interfere with fura-2 imaging at 340/380-nm excitation and 515-nm emission. GFP fluorescence was visualized using standard fluorescein isothiocyanate block. The two vectors were transfected using LipofectAMINE(+), according to the instructions of the manufacturer. Briefly, number 1 22-mm diameter round coverslips were seeded with AtT20 cells to low density (<30% confluence) and transfected in 1 ml of medium with the two vectors (0.8 μg of KSHV-GPCR and 0.2 μg of GFP DNAs/well). The coverslips were analyzed for calcium mobilization 48–72 h post-transfection, using the Life Sciences Miracal® system at 1–2 s/frame pair acquisition rate. Responses to 5 μmTRH were recorded at 37 °C. TRH, Acetylcholine, Groα, InsP3, and IP-10 were purchased from Sigma (Israel); GRP was from Bachem. 45CaCl2 was purchased from Amersham Pharmacia Biotech. Thapsigargin was purchased from Alomone Laboratories, Jerusalem. GFP Topaz® was from Packard. LipofectAMINE(+) was purchased from Life Technologies. Fura-2/AM was a product of Teflabs or Molecular Probes, Inc. (Eugene, OR). Ionomycin was from Calbiochem. All other chemicals were of analytical grade. ND96 solution was used in all short term incubations, and NDE solution was used for culturing of oocytes (21Asher O. Lupu-Meiri M. Jensen B.S. Paperna T. Fuchs S. Oron Y. FEBS Lett. 1998; 431: 411-414Crossref PubMed Scopus (11) Google Scholar). All experiments were performed on a large number of oocytes (n) from a number of donors (N). In calcium imaging experiments, several coverslips were assayed for each condition (N), and the number of cells was denoted byn. Results were presented as mean ± S.E. All results were analyzed by unpaired Student's t test withp < 0.05 considered as a significant difference between two populations. Expression of KSHV-GPCRs in Xenopus oocytes did not affect whole cell chloride currents or membrane potential in the absence of agonist. Moreover, there was no current response when the oocytes were exposed to several chemokine agonists (data not shown). However, expression of KSHV-GPCRs caused marked inhibition of Ca2+-activated chloride current responses to the cognate agonists of other coexpressed GPCRs. When 1–3 ng of KSHV-GPCR RNA was injected, the response to 1 μm TRH was inhibited from 2980 ± 148 to 576 ± 168 nA (81% inhibition,n = 145, N = 14) and the response to 10 μm acetylcholine (mediated by m1-Rs) was inhibited from 2067 ± 117 to 390 ± 44 (81% inhibition, n= 224, N = 22), and in a separate experiment in oocytes expressing the receptor for GRP, the response decreased from 2211 ± 426 to 675 ± 251 nA (70% inhibition, n = 11,N = 1). Representative responses to TRH are shown in Fig. 1 a and mean amplitudes in Fig. 1 b. The extent of inhibition was dose-dependent on the amount of KSHV-GPCR RNA injected (Fig. 2).Figure 2Inhibition of the response to TRH as a function of the amount of KSHV-GPCR RNA. Oocytes injected with RNA coding for TRH-Rs (1 ng/oocyte) alone or together with increasing amounts of RNA coding for KSHV-GPCRs (0.3–3.0 ng/oocyte) were assayed for responses to 1 μm TRH. A representative experiment (n = 15) is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We tested whether the apparent heterologous desensitization of responses to TRH in oocytes could be observed in mammalian cells. In human embryonic kidney HEK 293 EM cells, we included in our analysis only cells that were shown to respond to TRH. This most likely biased the analysis against showing an effect of coexpression of KSHV-GPCR, because 60% of the cells (n = 474, N = 5) in the population expressing TRH-Rs responded to TRH, whereas 47% of the cells (n = 430, N = 5) in the population expressing TRH-Rs and KSHV-GPCRs responded to TRH. It is likely, therefore, that some cells in the population expressing TRH-Rs and KSHV-GPCRs had no measurable response to TRH although they expressed TRH-Rs; i.e. KSHV-GPCR totally inhibited the response to TRH in these cells. These cells were not included in the analysis. HEK 293 EM cells transiently expressing TRH-Rs or TRH-Rs and KSHV-GPCRs exhibited similar "basal" [Ca2+]i; in TRH-R-expressing cells, basal [Ca2+]i was 82 ± 3 nm (n = 247, N= 3), and in TRH-R- and KSHV-GPCR-expressing cells, basal [Ca2+]i was 87 ± 4 nm(n = 201, N = 3). Typical responses to TRH are illustrated in Fig. 3. In TRH-R-expressing cells, there was a rapid increase in [Ca2+]i that peaked 12 s after TRH addition and declined rapidly, whereas in cells expressing TRH-Rs and KSHV-GPCRs there was a slower increase to a lower maximum followed by a slower decline. These time courses and amplitudes of responses are similar to those observed with control and desensitized TRH-Rs in HEK 293 cells (22Yu R. Hinkle P.M. J. Biol. Chem. 1997; 272: 28301-28307Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The peak increase in the fura signal was inhibited by 53 ± 2% in cells expressing TRH-Rs and KSHV-GPCRs compared with cells expressing TRH-Rs, corresponding to estimated increases in [Ca2+]i of approximately 400 and 200 nm, respectively. AtT20 pituitary cells stably expressing TRH-Rs (11Matus-Leibovitch N. Nussenzveig D.R. Gershengorn M.C. Oron Y. J. Biol. Chem. 1995; 270: 1041-1047Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) were transiently cotransfected with DNAs coding for KSHV-GPCR and for the Topaz mutant of GFP (see "Experimenal Procedures"). We assumed that cells transfected with GFP were likely to be also cotransfected with the KSHV-GPCR vector. Neighboring cells that displayed little or no GFP fluorescence were used as internal controls. In parallel, cells from the same batch, transfected with 0.2 μg of GFP-containing vector alone and exhibiting GFP fluorescence, were assayed as negative controls, reflecting the effect of GFP expression alone. In control cells, the net increase in [Ca2+]i was 646 ± 68 nm (9.2 ± 0.8-fold basal, n = 22, N = 5), while in cells expressing the viral receptor, the net increase in [Ca2+]i was only 301 ± 70 nm (4.9 ± 1.0-fold basal,n = 14, N = 5), i.e. 53% inhibition. By comparison, the response in cells transfected with GFP DNA alone was 504 ± 31 nm (9.4 ± 1.0-fold basal, n = 13, N = 3). We previously showed that a constitutively active TRH-R mutant causes homologous and heterologous desensitization of responses assayed inXenopus oocytes. This desensitization was fully or partially abolished by chlordiazepoxide, an inverse agonist of TRH-R (14Grimberg H. Zaltsman I. Lupu-Meiri M. Gershengorn M.C. Oron Y. Br. J. Pharmacol. 1999; 126: 1097-1106Crossref PubMed Scopus (11) Google Scholar). The heterologous desensitization caused by coexpression of KSHV-GPCRs with other receptors may therefore be attributed to the constitutive activity of the viral receptor. To test this hypothesis, we used IP-10, which was shown to act as an inverse agonist of KSHV-GPCRs in mammalian cells in culture (5Geras-Raaka E. Varma A. Ho H. Clark-Lewis I. Gershengorn M.C. J. Exp. Med. 1998; 188: 405-408Crossref PubMed Scopus (127) Google Scholar), to inhibit basal signaling. Incubation of oocytes coexpressing TRH-Rs and KSHV-GPCRs with 1 μm IP-10 resulted in a rapid increase in responses to TRH, i.e.resensitization of the response. Fig. 4illustrates the time course of resensitization of the response to TRH in a representative experiment. The results showed a rapid increase (within 10 min of the addition of IP-10), exhibiting a plateau around 8–9-fold the initial response at 40–80 min. This represented a resensitization of the response to TRH, from 6% of control in untreated oocytes to ∼35% at 60–80 min of incubation with IP-10. Similar resensitization was observed when oocytes coexpressing m1-Rs and KSHV-GPCRs were treated with IP-10 (not shown). While the degree of potentiation of the desensitized response (for either TRH-Rs or m1-Rs) following incubation with IP-10 was large (a 6.2 ± 2.3-fold increase, N = 11), the degree of resensitization varied among different experiments. In some experiments, only a moderate decrease in desensitization was observed, while in some a complete reversal was seen. Overall, incubation with 1 μm IP-10 for 30–80 min resulted in resensitization to a level of 49 ± 15% of control, compared with 18 ± 16% in oocytes not treated with the inverse agonist (n = 11). The results described here were consistent with the idea that KSHV-GPCR-induced desensitization may be caused by a high level of constitutive signaling by this receptor in Xenopus oocytes. It was therefore likely that persistent production of InsP3in oocytes resulted in constitutive Ca2+ mobilization affecting InsP3-sensitive Ca2+ stores. Indeed, oocytes coexpressing KSHV-GPCRs with TRH-Rs exhibited significantly higher rates of basal 45Ca2+ efflux than oocytes expressing TRH-Rs alone (Fig. 5). Expression of TRH-Rs did not affect the rates of basal45Ca2+ efflux either in control oocytes or in those coexpressing KSHV-GPCRs (data not shown). Moreover, the rate of entry of microinjected 45Ca2+ into a pool available for basal and agonist-induced release appeared to be much more rapid. In oocytes expressing KSHV-GPCRs, the maximal45Ca2+ release rate was reached within 42 ± 11 min after the injection of the radioactive tracer, while in control oocytes the maximal rate was achieved at ∼80 min (Fig. 5), confirming our previous results (13Shapira H. Lupu-Meiri M. Lipinsky D. Oron Y. Cell Calcium. 1996; 19: 201-210Crossref PubMed Scopus (8) Google Scholar). The maximal fractional rate of45Ca2+ efflux in oocytes (either naive or expressing TRH-Rs) was 0.19 ± 0.03% of residual label/min, and in those expressing KSHV-GPCRs it was 0.83 ± 0.12% of residual label/min (n = 22–25, N = 6) at 45–60 min after the injection of the label. IP-10 virtually abolished the enhanced basal 45Ca2+ efflux, thereby confirming its link to KSHV-GPCRs constitutive signaling activity. The addition of IP-10 abolished the enhanced efflux of45Ca2+ within 20 min (Fig.6).Figure 6IP-10 inhibits enhanced calcium efflux.Oocytes expressing KSHV-GPCRs were assayed for calcium efflux as described in Fig. 4. At 30 min of incubation (45 min after the injection of the label) 1 μm IP-10 was added to half of the cells assayed. The results are presented as fractional efflux rates. Each point represents the mean ± S.E. of eight oocytes. The vertical line indicates the time of addition of IP-10.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Oocytes expressing KSHV-GPCR responded to a challenge with the cytokine agonist Groα by a slow increase in 45Ca2+efflux. By 20 min of exposure to 1 μm of Groα, the fractional rate of efflux reached 1.14 ± 0.14, while the basal rate was 0.6 ± 0.06% of residual label/min (n = 18–19, N = 3). The increased rate of basal 45Ca2+ efflux caused by KSHV-GPCR was accompanied by a decrease in TRH-induced efflux in oocytes that coexpressed KSHV-GPCRs and TRH-Rs. While in oocytes expressing TRH-Rs alone, the net efflux of45Ca2+ induced by 15-min incubation with 10 μm TRH was 31 ± 5% of total label, coexpression of KSHV-GPCRs resulted in an efflux of 12 ± 4%, i.e.61% inhibition of the TRH response (Fig.7). This was also true for the initial phase of efflux within the first minute (data not shown), which is more directly related to the rapid, transient electrophysiological response to TRH. To investigate the mechanism of heterologous inhibition of TRH-R- and m1-R-mediated responses, we studied the effect of KSHV-GPCR expression on InsP3-evoked Ca2+ release by monitoring chloride current responses to microinjected InsP3. The injection of 0.92 pmol of InsP3 into control oocytes (injected with RNAs coding for TRH-Rs or m1-Rs) resulted in a response of 229 ± 16 nA (n = 103, N = 8), whereas responses of 91 ± 9 nA (n = 98,N = 8) were obtained in oocytes coexpressing KSHV-GPCRs. Hence, the expression of KSHV-GPCRs caused a 60% inhibition of the response to InsP3. When responses to agonists were compared with the responses to microinjected InsP3 in the same batches of oocytes, the responses to InsP3 were inhibited by 62% and those to agonists by 86%. These results suggest that the inhibition of InsP3-evoked calcium mobilization explains most of the marked inhibition of responses mediated by TRH-Rs or m1-Rs coexpressed with KSHV-GPCRs. IP-10 partially reversed the inhibition of responses to microinjected InsP3 (Fig. 8 A). Incubation of oocytes coexpressing TRH-Rs and KSHV-GPCRs with 1 μm IP-10 for 30–60 min increased the response to InsP3 from 26 to 51% of control. The time course of the action of IP-10 on InsP3-evoked responses (Fig.8 B) was similar to that on the TRH response (see Fig. 4) and on the enhanced efflux of 45Ca2+ (Fig. 6). To test the hypothesis that the constitutive signaling of KSHV-GPCR causes depletion of the calcium pool, we used thapsigargin, which inhibits the endoplasmic reticulum calcium ATPase and, consequently, empties the pool. Oocytes labeled with 45Ca2+were exposed to 2 μm thapsigargin. In control oocytes, the basal efflux was low, resulting in a cumulative loss of 16.6 ± 1.0% of total radiolabel at 150 min of incubation. The addition of thapsigargin at 30 min resulted in a major increase in efflux, leading to a loss of 83.5 ± 2.0% of total radiolabel. In contrast, oocytes expressing KSHV-GPCR exhibited a much higher efflux (loss of 75.0 ± 3.1% of total radiolabel at 150 min of incubation). Treatment with thapsigargin led to a small, statistically insignificant increase of cumulative efflux. After 120 min with thapsigargin, the KSHV-GPCR-expressing oocytes lost 76.0 ± 3.1% of total label. These results are shown in Fig.9 A. The effect of KSHV-GPCR's constitutive signaling on calcium pools was more apparent when fractional efflux rates were calculated (Fig. 9 B). Before the addition of thapsigargin, basal fractional efflux rates were 0.17 ± 0.03 and 0.76 ± 0.07% of residual label/min for control or KSHV-GPCR-expressing oocytes, respectively. Upon the addition of thapsigargin, the fractional efflux rate in control oocytes increased rapidly, to 1.43 ± 0.16% of residual label/min 30 min after the addition of the drug. Thereafter, the rate decreased slightly till the end of the incubation. In oocytes expressing KSHV-GPCRs, 20 min of incubation with thapsigargin accelerated the fractional efflux rate to a maximum of 1.84 ± 0.24% of residual label/min. Further incubation with thapsigargin led to a major decrease in fractional efflux rate, indicating that the depleted pools could not support the high rate of efflux. We previously showed that a constitutively signaling TRH-R mutant caused homologous (of TRH-R itself) and heterologous (of other coexpressed GPCRs) desensitization in Xenopus oocytes (11Matus-Leibovitch N. Nussenzveig D.R. Gershengorn M.C. Oron Y. J. Biol. Chem. 1995; 270: 1041-1047Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar,14Grimberg H. Zaltsman I. Lupu-Meiri M. Gershengorn M.C. Oron Y. Br. J. Pharmacol. 1999; 126: 1097-1106Crossref PubMed Scopus (11) Google Scholar). This desensitization was partially or fully abolished by short term incubation with chlordiazepoxide, an inverse agonist of TRH-R. KSHV-GPCR is known to signal constitutively in mammalian cells in culture, but its signaling properties in Xenopus oocytes have not been reported. The present study was designed to answer five questions relating to the biology of KSHV-GPCRs: Does KSHV-GPCR signal constitutively through the phosphoinositide-InsP3-calcium pathway in Xenopus oocytes? Can the signaling be abolished by an inverse agonist of KSHV-GPCR? Does KSHV-GPCR cause heterologous desensitization of other GPCRs when coexpressed in the same oocyte? What is the mechanism of desensitization? Does expression of KSHV-GPCR in mammalian cells result in heterologous desensitization? The data presented herein show that KSHV-GPCRs continuously stimulate calcium flux in oocytes. This finding is consistent with the idea that these viral receptors continuously stimulate InsP3generation and chronically affect cellular Ca2+ homeostasis and, therefore, signal constitutively in oocytes. This conclusion is supported by the finding that the specific inverse agonist IP-10 decreases calcium flux. Nevertheless, we observed no increased chloride current in oocytes expressing the viral receptor. This represents, most likely, a strong homologous desensitization of the response. The sluggish increase in 45Ca2+ efflux upon challenge with the KSHV-GPCR agonist, Groα, is compatible with the suggestion that the viral receptor's response is homologously desensitized. The same phenomenon was found, albeit to a lower level of stimulation, in oocytes and in HEK 293 EM cells (Fig. 3), mouse pituitary AtT20 cells expressing a constitutively active mutant of TRH-R, C335Stop (14Grimberg H. Zaltsman I. Lupu-Meiri M. Gershengorn M.C. Oron Y. Br. J. Pharmacol. 1999; 126: 1097-1106Crossref PubMed Scopus (11) Google Scholar). 2M. Lupu-Meiri and Y. Oron, unpublished observations. Our data clearly indicate that expression of KSHV-GPCRs causes pronounced (50–80%) desensitization of responses mediated by other GPCRs (TRH-Rs, m1-Rs, or GRP receptors) coexpressed in the same cell. In some experiments in oocytes, response inhibition greater than 99% was observed. This may have been true in HEK 293 EM cells also, because we found a smaller fraction of the cell population responding to TRH in populations expressing TRH-Rs and KSHV-GPCRs than in populations expressing TRH-Rs. Hence, the degree of heterologous desensitization attributable to KSHV-GPCRs greatly exceeded that caused by a constitutively active mutant TRH-R (14Grimberg H. Zaltsman I. Lupu-Meiri M. Gershengorn M.C. Oron Y. Br. J. Pharmacol. 1999; 126: 1097-1106Crossref PubMed Scopus (11) Google Scholar). To correlate the desensitization with the constitutive activity of KSHV-GPCR, we studied both basal and agonist-induced 45Ca2+ efflux as a reporter of cellular Ca2+ mobilization. Indeed, the basal fractional rate of 45Ca2+ efflux was much greater in oocytes expressing KSHV-GPCRs than in naive oocytes or those expressing other GPCRs. This enhancement of efflux was reflected both in the maximal fractional rates achieved (0.83 versus 0.19% of residual label/min) and in the time course of efflux development (reaching plateau twice as rapidly as in control oocytes). These results strongly suggest that the expression of KSHV-GPCRs is accompanied by a very rapid flux of Ca2+ through the releasable stores. Indeed, there is a direct correlation between the degree of heterologous desensitization caused by KSHV-GPCR (Fig.1 B) and the C335Stop mutant TRH-R (14Grimberg H. Zaltsman I. Lupu-Meiri M. Gershengorn M.C. Oron Y. Br. J. Pharmacol. 1999; 126: 1097-1106Crossref PubMed Scopus (11) Google Scholar) in Xenopusoocytes and the level of constitutive signaling activity observed in mammalian cells (2Arvanitakis L. Geras-Raaka E. Varma A. Gershengorn M.C. Cesarman E. Nature. 1997; 385: 347-350Crossref PubMed Scopus (577) Google Scholar, 23Heinflink M. Nussenzveig D.R. Grimberg H. Lupu-Meiri M. Oron Y. Gershengorn M.C. Mol. Endocrinol. 1995; 9: 1455-1460PubMed Google Scholar). Oocytes expressing KSHV-GPCRs exhibited impaired responses to InsP3 injection, suggesting that the viral receptor interferes with Ca2+ mobilization from the endoplasmic reticulum pool. The degree of inhibition of InsP3-evoked responses corresponded well with the degree of heterologous desensitization. The results observed in our system are consistent with depletion of InsP3-releasable Ca2+ pools. Indeed, experiments with thapsigargin clearly indicated that the size of releasable Ca2+ pool was much smaller in oocytes expressing KSHV-GPCRs. The finding that TRH-evoked45Ca2+ efflux is strongly inhibited in oocytes coexpressing TRH-Rs and KSHV-GPCRs is compatible with the heterologous desensitization of the electrophysiological response and with the data that suggest major depletion of calcium pool(s). Coexpression of KSHV-GPCRs and TRH-Rs in HEK 293 EM cells and AtT20 cells in culture resulted in 50% inhibition of TRH-evoked cytosolic calcium elevation, indicating that the viral receptor causes heterologous desensitization in mammalian cells as well. The heterologous desensitization in oocytes could have been a result of decreased expression of TRH-Rs (or, for that matter, any other GPCRs) due to coexpression of KSHV-GPCRs. There are two findings that argue against this interpretation. First, in the Xenopus oocyte system, coexpression of two GPCRs does not result in decreased expression and mutual response inhibition but rather in potentiation of responses mediated by either receptor (11Matus-Leibovitch N. Nussenzveig D.R. Gershengorn M.C. Oron Y. J. Biol. Chem. 1995; 270: 1041-1047Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Second, in mammalian cells, where receptor expression is easily quantified, there was no effect of cotransfection of KSHV-GPCR on the level of TRH-Rs expression under the conditions of these experiments. The findings presented here, together with our previous findings concerning a constitutively active TRH-R mutant (11Matus-Leibovitch N. Nussenzveig D.R. Gershengorn M.C. Oron Y. J. Biol. Chem. 1995; 270: 1041-1047Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 14Grimberg H. Zaltsman I. Lupu-Meiri M. Gershengorn M.C. Oron Y. Br. J. Pharmacol. 1999; 126: 1097-1106Crossref PubMed Scopus (11) Google Scholar, 23Heinflink M. Nussenzveig D.R. Grimberg H. Lupu-Meiri M. Oron Y. Gershengorn M.C. Mol. Endocrinol. 1995; 9: 1455-1460PubMed Google Scholar), strongly suggest that constitutively active receptors cause heterologous desensitization either in Xenopus oocytes or in mammalian cells. Considering the existence of pathologies resulting from mutations that cause constitutive activity and the findings that many wild type receptors exhibit some degree of constitutive activity, these novel desensitization phenomena should be further investigated. Last, our findings may have important implications for KSHV biology in infected cells expressing KSHV-GPCRs. Expression of KSHV-GPCRs leads to continuous stimulation of its signaling pathways while suppressing responses mediated by other GPCRs. Thus, in cells expressing KSHV-GPCRs, regulation of cell function has been coopted by a viral receptor, while the strong desensitization partially removes the infected cell from normal control by extracellular regulatory factors. These changes may create a cellular environment that is optimal for viral survival.