Protein Kinase C Phosphorylation of Threonine at Position 888 in Ca2+-Sensing Receptor (CaR) Inhibits Coupling to Ca2+ Store Release
1998; Elsevier BV; Volume: 273; Issue: 33 Linguagem: Inglês
10.1074/jbc.273.33.21267
ISSN1083-351X
AutoresMei Bai, Sunita Trivedi, Charles R. Lane, Yinhai Yang, Steven Quinn, Edward M. Brown,
Tópico(s)Magnesium in Health and Disease
ResumoPrevious studies in parathyroid cells, which express the G protein-coupled, extracellular calcium-sensing receptor (CaR), showed that activation of protein kinase C (PKC) blunts high extracellular calcium (Ca2+o)-evoked stimulation of phospholipase C and the associated increases in cytosolic calcium (Ca2+i), suggesting that PKC may directly modulate the coupling of the CaR to intracellular signaling systems. In this study, we examined the role of PKC in regulating the coupling of the CaR to Ca2+i dynamics in fura-2-loaded human embryonic kidney cells (HEK293 cells) transiently transfected with the human parathyroid CaR. We demonstrate that several PKC activators exert inhibitory effects on CaR-mediated increases in Ca2+i due to release of Ca2+ from intracellular stores. Consistent with the effect being mediated by activation of PKC, the inhibitory effect of PKC activators on Ca2+ release can be blocked by a PKC inhibitor. The use of site-directed mutagenesis reveals that threonine at amino acid position 888 is the major PKC site that mediates the inhibitory effect of PKC activators on Ca2+ mobilization. The effect of PKC activation can be maximally blocked by mutating three PKC sites (Thr888, Ser895, and Ser915) or all five PKC sites. In vitro phosphorylation shows that Thr888 is readily phosphorylated by PKC. Our results suggest that phosphorylation of the CaR is the molecular basis for the previously described effect of PKC activation on Ca2+o-evoked changes in Ca2+idynamics in parathyroid cells. Previous studies in parathyroid cells, which express the G protein-coupled, extracellular calcium-sensing receptor (CaR), showed that activation of protein kinase C (PKC) blunts high extracellular calcium (Ca2+o)-evoked stimulation of phospholipase C and the associated increases in cytosolic calcium (Ca2+i), suggesting that PKC may directly modulate the coupling of the CaR to intracellular signaling systems. In this study, we examined the role of PKC in regulating the coupling of the CaR to Ca2+i dynamics in fura-2-loaded human embryonic kidney cells (HEK293 cells) transiently transfected with the human parathyroid CaR. We demonstrate that several PKC activators exert inhibitory effects on CaR-mediated increases in Ca2+i due to release of Ca2+ from intracellular stores. Consistent with the effect being mediated by activation of PKC, the inhibitory effect of PKC activators on Ca2+ release can be blocked by a PKC inhibitor. The use of site-directed mutagenesis reveals that threonine at amino acid position 888 is the major PKC site that mediates the inhibitory effect of PKC activators on Ca2+ mobilization. The effect of PKC activation can be maximally blocked by mutating three PKC sites (Thr888, Ser895, and Ser915) or all five PKC sites. In vitro phosphorylation shows that Thr888 is readily phosphorylated by PKC. Our results suggest that phosphorylation of the CaR is the molecular basis for the previously described effect of PKC activation on Ca2+o-evoked changes in Ca2+idynamics in parathyroid cells. The extracellular calcium concentration (Ca2+o) is tightly regulated by the interactions of several hormones (e.g. parathyroid hormone (PTH), 1The abbreviations used are: PTHparathyroid hormoneCaRextracellular calcium-sensing receptorPKCprotein kinase CPMAphorbol myristate acetatePAGEpolyacrylamide gel electrophoresisMOPS4-morpholinepropanesulfonic acidbpbase pairsCa2+ oextracellular calcium. vitamin D, and calcitonin) and organ systems (i.e. parathyroid gland, kidney, bone, and intestine) (1Brown E.M. Physiol. Rev. 1991; 71: 371-411Crossref PubMed Scopus (637) Google Scholar). Parathyroid cells respond to changes in Ca2+o with oppositely directed alterations in PTH secretion through a cell surface, G protein-coupled receptor, the Ca2+o-sensing receptor (CaR). parathyroid hormone extracellular calcium-sensing receptor protein kinase C phorbol myristate acetate polyacrylamide gel electrophoresis 4-morpholinepropanesulfonic acid base pairs extracellular calcium. The CaR was first isolated from bovine parathyroid cells using expression cloning in Xenopus laevis oocytes and shows pharmacological properties nearly identical to those of the native receptor in its responses to agonists such as extracellular divalent cations (e.g. Ca2+o and Mg2+o), trivalent cations (e.g.Gd3+o) and polyamines (e.g. neomycin) (2Brown E.M. Gamba G. Riccardi D. Lombardi M. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2366) Google Scholar). In response to increases in Ca2+o, the CaR stimulates accumulation of inositol phosphates and produces transient followed by sustained increases in Ca2+i. Subsequently, cDNAs encoding the human homologue of the same receptor have been cloned from parathyroid (3Garrett J.E. Capuano I.V. Hammerland L.G. Hung B.C.P. Brown E.M. Hebert S.C. Nemeth E.F. Fuller F. J. Biol. Chem. 1995; 270: 12919-12925Abstract Full Text Full Text PDF PubMed Scopus (459) Google Scholar) and kidney (4Aida K. Koishi S. Inoue M. Nakazato M. Tawata M. Onaya T. J. Clin. Endocrinol. Metab. 1995; 80: 2594-2598Crossref PubMed Google Scholar) using a homology-based strategy. The physiological relevance of the CaR for mineral ion metabolism has been documented by the identification of CaR mutations in patients with inherited disorders of calcium homeostasis (36Pollak M.R. Brown E.M. Chou Y.H. Herbert S.C. Marx S.J. Steinmann B. Levi T. Seidman C.E. Seidman J.G. Cell. 1993; 75: 1297-1303Abstract Full Text PDF PubMed Scopus (908) Google Scholar, 37Pollak M.R. Brown E.M. Estep H.L. McLaine P.N. Kifor O. Park J. Herbert S.C. Seidman C.E. Seidman J.G. Nat. Genet. 1994; 8: 303-307Crossref PubMed Scopus (533) Google Scholar). High Ca2+o-evoked suppression of PTH secretion and the concurrent increases in Ca2+i in parathyroid cells can be negatively regulated by activation of protein kinase C (PKC) (5Clarke B.L. Hassager C. Fitzpatrick L.A. Endocrinology. 1993; 132: 1168-1175Crossref PubMed Scopus (20) Google Scholar, 6Kifor O. Congo D. Brown E.M. J. Bone Miner. Res. 1990; 5: 1003-1011Crossref PubMed Scopus (25) Google Scholar, 7Membreno L. Chen T.H. Woodley S. Gagucas R. Shoback D. Endocrinology. 1989; 124: 789-797Crossref PubMed Scopus (36) Google Scholar, 8Morrissey J.J. Am. J. Physiol. 1988; 254: E63-E70Crossref PubMed Google Scholar, 9Racke F.K. Nemeth E.F. J. Physiol. 1993; 468: 163-176Crossref PubMed Scopus (35) Google Scholar, 10Racke F.K. Nemeth E.F. J. Physiol. 1993; 468: 141-162Crossref PubMed Scopus (29) Google Scholar, 11Racke F.K. Nemeth E.F. Am. J. Physiol. 1994; 267 (E38): E429PubMed Google Scholar, 12Shoback D.M. Chen T.H. Endocrinology. 1990; 127: 141-148Crossref PubMed Scopus (20) Google Scholar, 13Watson P.H. Mortimer S.T. Tanguay K.E. Hanley D.A. J. Bone Miner. Res. 1992; 7: 667-674Crossref PubMed Scopus (10) Google Scholar). Such negative regulation by PKC has been suggested to be involved in the reduced responsiveness of adenomatous or hyperplastic parathyroid glands to Ca2+o as a result of an increase in membrane-associated PKC (14Ishizuka T. Kajita K. Kamikubo K. Komaki T. Miura K. Nagao S. Nozawa Y. Endocrinol. Jpn. 1987; 34: 965-968Crossref PubMed Scopus (25) Google Scholar, 15Ridefelt P. Nygren P. Hellman P. Larsson R. Rastad J. Akerstrom G. Gylfe E. Acta Endocrinol. 1992; 126: 505-509Crossref PubMed Google Scholar), although there is also reduced expression of the CaR in these glands (16Gogusev J. Duchambon P. Hory B. Giovannini M. Goureau Y. Sarfati E. Drüeke T.B. Kidney Int. 1997; 51: 328-336Abstract Full Text PDF PubMed Scopus (439) Google Scholar, 17Kifor O. Moore Jr., F.D. Wang P. Goldstein M. Vassilev P. Kifor I. Hebert S.C. Brown E.M. J. Clin. Endocrinol. Metab. 1996; 81: 1598-1606Crossref PubMed Google Scholar). Likewise, PKC may contribute to age-related changes in the regulation of PTH secretion by Ca2+o in rats (18Wongsurawat N. Armbrecht H.J. Exp. Gerontol. 1987; 22: 263-269Crossref PubMed Scopus (16) Google Scholar). Therefore, stimulus-secretion coupling in parathyroid cells can be modulated by PKC, perhaps at an early step in the process of Ca2+o sensing. The human homologue of the CaR is predicted to have five PKC sites in its intracellular domains. We hypothesized that PKC modulates the sensitivity of parathyroid cells to changes in Ca2+o by covalently modifying these sites. To test this hypothesis, we have transiently transfected a human parathyroid CaR cDNA (18Wongsurawat N. Armbrecht H.J. Exp. Gerontol. 1987; 22: 263-269Crossref PubMed Scopus (16) Google Scholar) in HEK293 cells and mutated each of the five putative PKC sites individually or in varying combinations in the CaR. We studied Ca2+i responses of the wild type and mutant CaRs to elevations in Ca2+o and the polycationic CaR agonist, neomycin, in the presence or absence of various PKC activators (e.g. phorbol myristate acetate (PMA), Mezerein, and (−)-Indolactam V) and/or the PKC inhibitor, staurosporine. Our results show that phosphorylation by PKC at one of the five predicted PKC phosphorylation sites (Thr888) substantially reduces CaR-mediated release of Ca2+ from intracellular stores. Therefore, it is possible that PKC phosphorylation of the CaR regulates PTH secretion by inhibiting Ca2+ mobilization or perhaps generation of some other intracellular mediator(s) along the inositol trisphosphate/phospholipase C pathway. Site-directed mutagenesis was performed using the approach described by Kunkel (19Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4900) Google Scholar) to produce mutated receptors in which one or more serine or threonine residues present in the five predicted PKC phosphorylation sites within intracellular domains of the human CaR were mutated to alanine or valine, respectively. The dut-1 ung-1 strain of Escherichia coli, CJ236, was transformed separately with mutagenesis cassette 5 or 6, as described previously (20Bai M. Quinn S. Trivedi S. Kifor O. Pearce S.H.S. Pollak M.R. Krapcho K. Hebert S.C. Brown E.M. J. Biol. Chem. 1996; 271: 19537-19545Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). For two receptors with two mutations each (T888V/S895A and T888V/S915A) and one with three mutations (T888V/S895A/S915A), CJ236 cells were separately transformed with a mutated cassette 6 carrying a single mutation (S895A or T888V) or two mutations (T888V/S915A). Uracil-containing, single-stranded DNA was produced by infecting the cells with the helper phage, VCSM13 (Stratagene, La Jolla, CA). The single-stranded DNA was then annealed to a mutagenesis primer that contained the desired nucleotide change encoding a single point mutation flanked on both sides by wild type sequences. The primer was subsequently extended around the entire single-stranded DNA and ligated to generate closed circular heteroduplex DNA. DH5α competent cells were transformed with these DNA heteroduplexes, and incorporation of the desired mutations was confirmed by sequencing the entire cassettes. The resultant mutated cassette 5 was doubly digested with HpaI and XhoI and cloned into the reconstructed receptor in pcDNA3 (Invitrogen), as described previously (20Bai M. Quinn S. Trivedi S. Kifor O. Pearce S.H.S. Pollak M.R. Krapcho K. Hebert S.C. Brown E.M. J. Biol. Chem. 1996; 271: 19537-19545Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). Likewise, mutated versions of cassette 6 containing the desired mutations were doubly digested withXhoI and XbaI and cloned into the reconstructed receptor in pcDNA3. Cassette 6 was doubly digested with XhoI and HhaI, and the same cassette carrying three mutated PKC sites was doubly digested with HhaI and XbaI. Two small fragments (404 and 568 bp) obtained from the above digestions were ligated to the large fragment resulting from digestion of the parent reconstructed CaR clone with XhoI andXbaI. The resultant clone was confirmed by sequencing. The mutant receptor carrying T646V was doubly digested with HpaI and XhoI, and the mutant receptor carrying S794A was doubly digested with XhoI andXbaI. The two fragments (420 and 975 bp) obtained form the above digestions were ligated to the large fragment resulting from digestion of the wild type CaR in pcDNA3 with HpaI andXbaI. The resultant clone was confirmed by sequencing to carry these two mutations. Cassette 6 carrying S794A was doubly digested withXhoI and SphI, and the same cassette carrying three mutations was doubly digested with SphI andXbaI. Two fragments (168 and 795 bp) obtained from the above digestions were ligated to the large fragment resulting from digestion of the CaR carrying the single mutation, T646V, with XhoI and XbaI. The resultant clone was confirmed by sequencing to carry all five mutations. The receptors carrying five mutated PKC sites and two mutated sites (S895A/S915A) was doubly digested with KpnI andXbaI to obtain the full-length CaR inserts, which were further digested with SphI. One fragment (2434 bp) containing T646V/S794A and another fragment (803 bp) containing S895A/S915A, obtained from the above digestions, were ligated to pcDNA3 generated by KpnI and XbaI. The resultant clone was confirmed by sequencing to carry four mutations. The Flag, an epitope tag, was introduced into the third cassette of the wild type CaR as described previously (21Bai M. Janicic N. Trivedi S. Quinn S.J. Cole D.E.C. Brown E.M. Hendy G.N. J. Clin. Invest. 1997; 99: 1917-1925Crossref PubMed Scopus (78) Google Scholar). The third cassette containing Flag was digested with AflII and NheI and ligated to the large fragments resulting from digestion of the CaRs containing PKC site mutations. CaR cDNAs were prepared using the Midi Plasmid Kit (Qiagen). LipofectAMINE (Life Technologies, Inc.) was employed as a DNA carrier for transfection (22Hawley-Nelson P. Ciccarone V. Gebeyehu G. Jessee J. Felgner P.L. Focus. 1993; 15: 73-79Google Scholar). The HEK293 cells used for transient transfection were provided by NPS Pharmaceuticals, Inc. (Salt Lake City, UT) and were cultured in DMEM (Life Technologies, Inc.) with 10% fetal bovine serum (Hyclone). The DNA-liposome complex was prepared by mixing DNA and LipofectAMINE in Opti-MEM I reduced serum medium (Life Technologies, Inc.) and incubating the mixture at room temperature for 30 min. The DNA-LipofectAMINE mixture was then diluted with Opti-MEM I reduced serum medium and added to 90% confluent HEK293 cells plated on 13.5 × 20.1-mm glass coverslips using 0.625 μg of DNA (for measurement of Ca2+i) or in 100 mm Petri dishes using 3.75 μg of DNA (for obtaining protein for Western analysis). After 5 h of incubation at 37 °C, equivalent amounts of Opti-MEM I reduced serum medium with 20% fetal bovine serum were added to the medium overlying the transfected cells, and the latter was replaced with fresh DMEM with 10% fetal bovine serum at 24 h after transfection. The expressed Ca2+o-sensing receptor protein was assayed 48 h after the start of transfection. HEK293 cells, which were plated on coverslips and transfected with CaR cDNAs, were loaded for 2 h at room temperature with fura-2/AM (Molecular Probes) in 20 mmHEPES, pH 7.4, containing 125 mm NaCl, 4 mmKCl, 1.25 mm CaCl2, 1 mmMgSO4, 1 mm NaH2PO4, 0.1% (w/v) bovine serum albumin, and 0.1% dextrose and washed once at 37 °C for 20–30 min with a buffer solution (20 mmHEPES, pH 7.4, containing 125 mm NaCl, 4 mmKCl, 0.5 mm CaCl2, 0.5 mmMgCl2, 0.1% dextrose, and 0.1% bovine serum albumin). The coverslips were then placed diagonally in a thermostatted quartz cuvette containing the buffer solution, using a modification of the technique employed previously in this laboratory (23Fajtova V.T. Quinn S.J. Brown E.M. Am. J. Physiol. 1991; 261: E151-E158PubMed Google Scholar). The CaR was activated by multiple additions of an agonist in incremental doses to reach the desired concentrations. Excitation monochrometers were centered at 340 and 380 nm with emission light collected at 510 ± 40 nm through a wide-band emission filter. The ratio of emitted light (340/380 excitation) was used as readout for Ca2+ias described previously (23Fajtova V.T. Quinn S.J. Brown E.M. Am. J. Physiol. 1991; 261: E151-E158PubMed Google Scholar). For PKC activation, the cells were preincubated with the PKC activators (PMA, mezerein, or (−)-indolactam V) for 1–2 min. For PKC inhibition, the cells were preincubated with staurosporine for 30 min. To measure transient Ca2+i responses elicited by neomycin, the buffer solution was devoid of MgCl2, CaCl2, and bovine serum albumin, and 1 mm EGTA was added at the beginning of the experiment. To evaluate the activities of the wild type and mutant receptors, the cumulative Ca2+i response at a given concentration of the agonist was determined using the following method. If the peak increases in Ca2+i are P1,P2, P3 … Pn at concentrations of the agonist in the bath solution corresponding to C1,C2, C3 … Cn, which were achieved by incremental additions of the agonist, the cumulative Ca2+i response (Rn) at any given agonist concentration (Cn) is defined as the sum, P1 +P2 + P3+ … +Pn. The responsiveness of the wild type and mutant receptors to agonists were compared by determining both EC50 values and the maximal responses of the respective CaRs. EC50 has been defined as the effective concentration of an agonist giving half of the maximal Ca2+i response and was determined by plotting the concentration-response curve. The cumulative maximal Ca2+i response has been defined as the cumulative Ca2+i response at the highest agonist concentration achieved by the last addition. The mean EC50 for the wild type or each mutant receptor in response to increasing concentrations of Ca2+o or other CaR agonists was calculated from the EC50 values for all of the individual experiments and is expressed with the S.E. as the index of dispersion. Comparisons of the EC50 values were performed using analysis of variance or Duncan's multiple comparison test (24Duncan D.B. Biometrics. 1955; 11: 1-42Crossref Google Scholar). A p value of ≤0.05 was considered to indicate a statistically significant difference. Crude plasma membranes were isolated from HEK293 cells transiently transfected with the wild type or mutant receptors by differential speed centrifugation as described by Sun et al. (25Sun G.Y. Huang H.-M. Kelleher J.A. Stubbs E.B.J. Sun A.Y. Neurochem. Int. 1988; 12: 69-77Crossref PubMed Scopus (60) Google Scholar). Confluent cultured cells in 100-mm culture plates were rinsed twice with phosphate-buffered saline and treated with 0.02% EDTA in phosphate-buffered saline at 37 °C for 5 min. The detached cells were pelleted and suspended in 300 μl of homogenization buffer: 50 mm Tris-HCl, pH 7.4, containing 0.32 m sucrose, 2 mm EDTA, and a mixture of protease inhibitors (83 μg/ml aprotinin, 30 μg/ml leupeptin, 1 mg/ml Pefabloc, 50 μg/ml calpain inhibitor, 50 μg/ml bestatin, and 5 μg/ml pepstatin (Boehringer Mannheim)). Then the cells were homogenized with 15 strokes of a motor-driven Teflon pestle in a tightly fitting glass tube. The homogenate was sedimented at 18,800 × g for 20 min to remove nuclei and mitochondria. The supernatant was subsequently sedimented at 43,500 × g for 20 min to pellet the plasma membranes, and the resultant pellet was solubilized with 1% Triton X-100. All steps were carried out at 4 °C. After determination of protein concentrations in the crude plasma membrane preparations using the Pierce BCA protein assay, an appropriate amount of membrane protein (4 μg) was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (26Laemmli U.K. Nature. 1970; 227(259): 680-685Crossref Scopus (207231) Google Scholar). The proteins on the gel were subsequently electrotransferred to a nitrocellulose membrane. After being blocked with 5% milk, the blot was incubated with a previously characterized primary anti-CaR antibody (4641) (20Bai M. Quinn S. Trivedi S. Kifor O. Pearce S.H.S. Pollak M.R. Krapcho K. Hebert S.C. Brown E.M. J. Biol. Chem. 1996; 271: 19537-19545Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar) and then with a secondary, goat anti-rabbit antibody conjugated to horseradish peroxidase (Sigma, diluted 1:500). The Ca2+o-sensing receptor protein was detected with an ECL system (Amersham Pharmacia Biotech). HEK293 cells transiently transfected with receptors were rinsed twice with phosphate-buffered saline and solubilized with 1% Triton X-100, 0.5% Nonidet P-40, 150 mm NaCl, 10 mm Tris, pH 7.4, 2 mm EDTA, 1 mm EGTA, protease inhibitors, including 83 μg/ml aprotinin, 30 μg/ml leupeptin, 1 mg/ml Pefabloc, 50 μg/ml calpain inhibitor, 50 μg/ml bestatin, and 5 μg/ml pepstatin (1× immunoprecipitation buffer), at room temperature. Insoluble materials were removed by centrifuging the cell lysates at 15,000 rpm for 15 min at 4 °C. The supernatants were collected as total cell lysates. The protein concentration was determined using the Pierce BCA protein assay. To a microcentrifuge tube, 5 μg of monoclonal anti-Flag M2 antibody (VWR Scientific), 400 μl of H2O, 500 μl of 2× immunoprecipitation buffer, and 100 μl of total lysate containing 500 μg of protein were added. The mixture was incubated at 4 °C for 1 h. To the mixture was added 5 μl of an alkaline phosphatase-conjugated, anti-mouse IgG. The incubation was continued for an additional 30 min at 4 °C. To the mixture was then added 50 μl of 10% protein A-agarose (Life Technologies, Inc.) for an additional 3-h incubation at 4 °C. The immunoprecipitates were washed three times with 1× immunoprecipitation buffer and twice with phosphate-buffered saline containing protease inhibitors as described above. After one additional wash with 50 μl of PKC assay dilution buffer (20 mm MOPS, pH 7.2, 25 mm β-glycerol phosphate, 1 mm sodium orthovanadate, 1 mm dithiothreitol, 1 mmCaCl2) purchased from Upstate Biotechnology, the samples were ready for in vitro phosphorylation. The samples were phosphorylated with 80 ng of PKC (Upstate Biotechnology) in 50 μl of 20 mm MOPS, pH 7.2, containing 25 mmβ-glycerol phosphate, 1 mm sodium orthovanadate, 1 mm dithiothreitol, 1 mm CaCl2, 100 μm (20 μCi) [γ-32P]ATP, 15 mm MgCl2, 0.1 mg/ml phosphatidylserine, 0.01 mg/ml diglyceride, 0.4 μm protein kinase A inhibitor peptide, 4 μm compound R24571 (an inhibitor for calmodulin-dependent kinases). The samples were incubated for 10 min at 32 °C and washed three times with a washing buffer (10 mm Tris-HCl, pH 7.4, 75 mm NaF, 20 mm β-glycerol phosphate, 0.1% Triton X-100). The pellet was extracted with 45 μl of 2× SDS sample buffer at 65 °C for 30 min. One-third of the eluted sample was subjected to SDS-PAGE and electrotransferred to a nitrocellulose membrane. Phosphorylated protein bands were visualized by autoradiography. The CaR immunoreactivities in the samples were determined by Western analysis. To examine the effects of PKC activators on the CaR, transiently transfected HEK293 cells were treated for 1–3 min with the vehicle (Me2SO), PKC activators (PMA, mezerein, or (−)-indolactam V), or an inactive phorbol analogue (4α-phorbol 12,13-didecanoate, used as a negative control) prior to activation of the CaR by elevating Ca2+o. The activity of the receptor was evaluated by measuring EC50 and maximal cumulative response (see under “Experimental Procedures” for definition). Cells treated with Me2SO or 1 μm inactive phorbol derivative responded to increasing concentrations of Ca2+o in a manner similar to untreated cells, with EC50[Ca2+o] values of 4.1 ± 0.1 (n = 7), 4.0 ± 0.1 (n = 8), and 4.0 ± 0.1 (n = 34) mm, respectively, without significant differences (p ≤ 0.05). A representative tracing of the control Ca2+iresponses is shown in Fig. 1 A. In cells treated with 100 nm PMA, the Ca2+i responses at low Ca2+oconcentrations (1.5–4.5 mm) were markedly attenuated (Fig.1 B). At higher concentrations of Ca2+o(5.5 mm and above), however, the Ca2+o-elicited increases in Ca2+i were similar to those of control cells. As a result, the cumulative maximal Ca2+i response to elevated Ca2+o was reduced to 41% of the control by PMA, with a significant increase in EC50[Ca2+o] to 5.0 ± 0.1 mm (n = 25) (p ≤ 0.05). Additional, structurally unrelated PKC activators, such as 1 μm mezerein and 500 nm (−)-indolactam V had essentially identical inhibitory effects on CaR-elicited Ca2+i responses (Fig.2).Figure 2Inhibitory effects of structurally unrelated PKC activators on Ca2+o-elicited Ca2+i responses of the CaR. HEK293 cells were transfected with the CaR and loaded with fura-2. Changes in the emission ratio (340/380 excitation) were measured to assess Ca2+o-evoked Ca2+i responses. Prior to additions of Ca2+o, the cells were treated or not treated with one of the PKC activators, PMA, Mezerein, or (−)-Indolactam V. Responses are normalized to the maximal cumulative Ca2+i responses of nontreated cells. Eachpoint is the mean value of the number of measurements indicated in parentheses. S.E. values are indicated withvertical bars through each point. Some error barsare smaller than the symbol.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because release of Ca2+ from intracellular stores as well as Ca2+ influx could contribute to increases in Ca2+i when Ca2+o is used to activate the CaR, it is essential to use other CaR agonists, such as neomycin, to determine the impact of PKC activators on CaR-evoked mobilization of intracellular Ca2+ in the absence of Ca2+o. The CaR was activated by neomycin in the absence of Ca2+o and Mg2+o, as well as in the presence of 1 mm EGTA. Therefore, the neomycin-elicited Ca2+i responses in cells transfected with the CaR (Fig. 1 C) were solely the result of release of Ca2+ from intracellular stores, and these responses were not present in cells mock-transfected with vector alone (data not shown). Neomycin-elicited Ca2+i responses were substantially attenuated by pretreatment with PMA (1 μm) (Fig. 1 D) at all concentrations of neomycin tested, and the maximal cumulative Ca2+iresponse was reduced to 25% of the control. PMA treatment increased the EC50[neomycin] of the CaR from 298 ± 9 μm (n = 8) to 405 ± 22 μm (n = 8). The marked difference in the maximal cumulative responses in PMA-treated cells stimulated with Ca2+o versus neomycin in the absence of Ca2+o (64 and 25%, respectively) suggested that calcium influx stimulated by CaR agonists might be less affected by PMA than calcium mobilization from intracellular stores. To evaluate the effect of PMA on high Ca2+o-evoked sustained increases in Ca2+i as an indirect assessment of Ca2+ influx, Ca2+o-elicited transient increases in Ca2+i were permitted to fall for a longer period of time (100 s in Fig. 3 versus25 s in Fig. 1) after each addition of Ca2+o. The sustained responses observed in this experiment were defined as the increased levels of Ca2+i that remained at 100 s after each incremental addition of agonist, and these responses reflect the new steady states, in which influx and efflux of Ca2+ are nearly equal. As shown in Fig. 3, PMA markedly reduced the transient Ca2+i responses at low Ca2+o concentrations (1.5–3.5 mm), similar to the earlier observations made in Fig. 1, A andB. Thus the cumulative transient Ca2+iresponses were markedly affected by PMA (Fig.4 A). In contrast, the sustained Ca2+i responses were similar in control and PMA-treated cells (Fig. 4 B). In cells mock-transfected with vector alone, there was also a gradual Ca2+iincrease. However, at least 60% of this increase resulted from leakage of fura-2 over the ∼20-min time course of these experiments, which was marginally affected by PMA (Fig. 3, C and D, and Fig. 4). Although the transfection efficiency was less than 25%, the sustained Ca2+i responses in the receptor-transfected cells were significantly higher than those in vector-transfected cells.Figure 4Concentration dependence for the Ca2+o-evoked transient and sustained Ca2+i responses in the presence or absence of PMA. In this figure, the transient and sustained Ca2+i responses in Fig. 3 were plotted against the concentrations of Ca2+o added (see under “Experimental Procedures”). HEK293 cells transfected with the pcDNA3 vector (circles), or the CaR (squares) were mock-treated (open symbols) or treated (filled symbols) with PMA. A, all of the transient responses (i.e. the cumulative peak responses) are normalized to the maximal cumulative response of the CaR at 20 mmCa2+o in the absence of PMA. B, all of the sustained responses (i.e. the increased level of Ca2+i that persisted at 100 s after each addition of Ca2+o) are normalized to the maximal sustained response of the CaR at 20 mmCa2+o in the absence of PMA. Each pointis the mean value of the number of measurements indicated in parentheses. S.E. values are indicated with vertical barsthrough each point. Some error bars are smaller than the symbol.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In order to demonstrate further that the effect of PKC activators on the function of the CaR was mediated by PKC, we examined the effect of a PKC inhibitor, staurosporine, on CaR-evoked Ca2+iresponses (Fig. 5). Pretreatment of CaR-transfected HEK293 cells with 1 μm staurosporine for 30 min significantly reduced the EC50[Ca2+o] from 4.0 ± 0.1 mm (n = 34; TableI) to 2.9 ± 0.1 mm(n = 22; Table I) (p ≤ 0.05). In addition, the pretreatment prevented the inhibitory effects of PMA (Fig. 5) and other PKC activators (data not shown) on the Ca2+i responses to the same extent, further supporting the conclusion that the effect of PKC activators on CaR-dependent changes in
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