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Originally published In Press as doi:10.1074/jbc.M607469200 on March 21, 2007

J. Biol. Chem., Vol. 282, Issue 20, 15048-15056, May 18, 2007
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Protein Kinase C-mediated Phosphorylation of the Calcium-sensing Receptor Is Stimulated by Receptor Activation and Attenuated by Calyculin-sensitive Phosphatase Activity*

Sarah L. Davies{ddagger}, Ai Ozawa{ddagger}, Wanda D. McCormick{ddagger}, Melita M. Dvorak{ddagger}§, and Donald T. Ward, Recipient of Kidney Research UK (NKRF) Career Development Fellowship TF6/2002{ddagger}1

From the {ddagger}Faculty of Life Sciences, University of Manchester, Manchester M13 9PL, United Kingdom and the §Department of Endocrinology, University of California, San Francisco, California 94121

Received for publication, August 7, 2006 , and in revised form, January 17, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The agonist sensitivity of the calcium-sensing receptor (CaR) can be altered by protein kinase C (PKC), with CaR residue Thr888 contributing significantly to this effect. To determine whether CaRT888 is a substrate for PKC and whether receptor activation modulates such phosphorylation, a phospho-specific antibody against this residue was raised (CaRpT888). In HEK-293 cells stably expressing CaR (CaR-HEK), but not in cells expressing the mutant receptor CaRT888A, phorbol ester (PMA) treatment increased CaRpT888 immunoreactivity as observed by immunoblotting and immunofluorescence. Raising extracellular Ca2+ concentration from 0.5 to 2.5 mM increased CaRT888 phosphorylation, an effect that was potentiated stereoselectively by the calcimimetic NPS R-467. These responses were mimicked by 5 mM extracellular Ca2+ and abolished by the calcilytic NPS-89636 and also by PKC inhibition or chronic PMA pretreatment. Whereas CaRT888A did exhibit increased apparent agonist sensitivity, by converting intracellular Ca2+ (Ca2+i) oscillations to sustained plateau responses in some cells, we still observed Ca2+i oscillations in a significant number of cells. This suggests that CaRT888 contributes significantly to CaR regulation but is not the exclusive determinant of CaR-induced Ca2+i oscillations. Finally, dephosphorylation of CaRT888 was blocked by the protein phosphatase 1/2A inhibitor calyculin, a treatment that also inhibited Ca2+i oscillations. In addition, calyculin/PMA cotreatment increased CaRT888 phosphorylation in bovine parathyroid cells. Therefore, CaRT888 is a substrate for receptor-induced, PKC-mediated feedback phosphorylation and can be dephosphorylated by a calyculin-sensitive phosphatase.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The extracellular calcium-sensing receptor (CaR)2 is a type III G protein-coupled receptor, whose primary function is to regulate parathyroid hormone (PTH) secretion and thus whole body calcium homeostasis (1, 2). The parathyroid CaR acts by responding to elevated extracellular Ca2+ (Ca2+o) concentration in the blood to mobilize Ca2+i release, as well as other intracellular signals to suppress PTH production and release. In dispersed bovine parathyroid cells, the increase in Ca2+i concentration and suppression of PTH secretion elicited by high Ca2+o concentration can be inhibited by phorbol ester treatment (3-5).

The human CaR contains five predicted PKC consensus sites, two within its intracellular loops (Thr646 and Ser794) and three in its intracellular domain (Thr888, Ser895, and Ser915) (6). Mutation of the two intracellular loop residues had little effect on Ca2+o sensitivity, whereas mutants T888V, S895A, and S915A each exhibit increased CaR sensitivity for Ca2+o, the most dramatic effect being with the mutation at Thr888 (7). Substitution of Thr888 with negatively charged amino acids mimics the effect of phorbol ester treatment on wild-type CaR (8). There is also evidence that CaRT888A elicits sustained Ca2+i mobilization rather than oscillations (9).

Whereas this suggests that PKC-mediated CaR phosphorylation regulates CaR function, no reagents have existed previously for investigating CaR phosphorylation directly. Thus, we have developed a phospho-specific antibody that recognizes the phosphorylated form of CaRT888, this residue being chosen because its mutation elicited the greatest effect on CaR function of the putative PKC sites.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The calcimimetics NPS R-467 and S-467 and the calcilytic NPS-89636 were a kind gift of Dr. E. F. Nemeth and NPS Pharmaceuticals, Inc. (Toronto, Canada). Fura-2/AM was from Invitrogen and Gö6976, PKCbeta-selective inhibitor (anilinomonoindolylmaleimide), and endothall thioanhydride (7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid) were from Calbiochem (EMD Biosciences, Inc., San Diego, CA). Horse-radish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies were from DakoCytomation (Ely, Cambridgeshire, UK). Unless stated otherwise, all other chemicals were purchased from Sigma.

Cell Culture—HEK293 cells, stably transfected with human parathyroid CaR (6), were a gift from Dr. E. F. Nemeth (NPS Pharmaceuticals). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen Ltd., Paisley, Scotland, UK) and 200 µg/ml hygromycin B (Roche Applied Science).


Figure 1
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  		FIGURE 1.
CaRT888 phosphorylation in CaR-HEK cells. A, CaR-HEK cells were incubated with PMA (1 µM, 10 min), lysed, and solubilized in Laemmli buffer and then processed for immunoblotting using either ADD monoclonal antibody (total CaR) or anti-CaRpT888 antibody as stated. In A, panel ii, solubilization occurred either under non-reducing (NR) or reducing (beta-mercaptoethanol, betaMe) conditions. In A, panel iii, the CaRpT888 antibody was used to immunoprecipitate the fraction of CaR proteins phosphorylated on residue Thr888 with the resulting immunoprecipitate pellets and supernatants immunoblotted using the total CaR antibody. Where indicated, some cells were cotreated with PMA (1 µM) and calyculin (100 nM) prior to lysis. B, cells, preincubated for 10 min in the absence or presence of PMA, were processed for immunoblotting (i) or immunofluorescence (ii) using anti-CaRpT888 antibody preincubated in the absence or presence of phosphorylated (+phos) or nonphosphorylated (+nonphos) immunizing peptide. All data are representative of a minimum of three independent experiments.

 
Phosphorylation Assay—Cells were grown to 80-90% confluence in 35-mm culture dishes and assayed as described previously (10, 11). Briefly, cells were rinsed in phosphate-buffered saline for 5 min and equilibrated for 20 min in Experimental Buffer ((mM) 20 HEPES (pH 7.4), 125 NaCl, 4 KCl, 0.5 CaCl2, 0.5 MgCl2, and 5.5 glucose) at 37 °C. Cells were then exposed to various experimental treatments for 10 min and then lysed on ice in RIPA buffer (12 mM HEPES (pH 7.6), 300 mM mannitol, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, 1.25 µM pepstatin, 4 µM leupeptin, 4.8 µM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 mM EGTA, 100 µM vanadate, 1 mM NaF, and 250 µM sodium pyrophosphate). In all experiments where CaCl2 concentration was increased, the concentration of NaCl was reduced accordingly to normalize osmolarity. Finally, cell lysate was then mixed with 5x Laemmli buffer and heated at 65 °C for 3 min prior to immunoblotting.

Immunoblotting and Immunocytochemistry—Immunoblotting was performed as described previously (10, 11). Anti-CaR mouse monoclonal antibody, raised to amino acids 214-235 (ADD) of the extracellular domain of the human parathyroid CaR was from Affinity Bioreagents (Golden, CO) and anti-protein phosphatase 2A (catalytic subunit) monoclonal antibody was from Upstate (Millipore, Chandlers Ford, Hampshire, UK). Phosphorylation of CaR residue Thr888 was studied using a custom-generated (Sigma Genosys) polyclonal antibody raised to the phosphorylated form of Thr888 contained within amino acids 882-896 of the human CaR sequence (KVAARA(pT) LRRSNVSR). The antibody was affinity purified using the nonphosphorylated peptide to remove non-phosphospecific antibody, followed by collection of the phosphospecific antibody (1.1 mg/ml) on a column prepared using the phosphorylated peptide. For immunoprecipitation experiments, cell lysates prepared using SDS-free RIPA buffer were precleared with Protein A-Sepharose, then mixed with anti-CaRpT888 antibody (1:100) followed by Protein A-Sepharose overnight (4 °C with constant rotation). The immunoprecipitates were collected by centrifugation and washed 3 times in SDS-free RIPA buffer prior to immunoblotting as before.

For immunofluorescence, cells were grown on coverslips to 50-80% confluence and fixed with 10% (w/v) paraformaldehyde solution at room temperature for 30 min and permeabilized with 0.075% (w/v) saponin in phosphate-buffered saline for 10 min. Indirect immunofluorescence was performed using the anti-CaRpT888 polyclonal antibody (1:200 dilution) and an Alexa 488-conjugated goat anti-rabbit secondary antibody (1:1000; Molecular Probes). Alternatively, anti-CaR monoclonal (ADD; 1:200) was used with a donkey anti-mouse secondary antibody conjugated with Alexa 594 (1:1000; Molecular Probes). In some experiments the primary antibody was preincubated overnight at 4 °C with an excess of antigenic peptide (either phosphorylated or non-phosphorylated) prior to incubation with the cells. Immunofluorescence was examined using a Zeiss Axioplan 2 fluorescence microscope with images acquired using a Hamamatsu digital camera, with each sample imaged under identical exposure conditions. Images were processed using the software package KS300 version 3.0 (Carl Zeiss Ltd., Hertfordshire, UK).

Intracellular Ca2+ Assay—CaR-HEK cells were cultured on glass coverslips and loaded with Fura-2/AM (1 µM for 1 h) at room temperature in the dark in Ca2+ assay buffer (20 mM HEPES, pH 7.4, 125 mM NaCl, 4 mM KCl, 1.2 mM CaCl2, 0.5 mM MgCl2, 5.5 mM glucose) supplemented with 0.1% bovine serum albumin. Non-absorbed Fura-2/AM was removed by washing and the cells were equilibrated for 10 min in Experimental Buffer containing the baseline [Ca2+]o appropriate for the ensuing experiment. The cells were mounted in a perfusion chamber (Warner Instruments, Hamden, CT) and observed through a x40 oil-immersion objective. Dual-excitation wave-length microfluorometry was then performed using a Nikon Diaphot inverted microscope (Cairn Research Ltd., Kent, UK). Experiments were performed at room temperature in Ca2+ assay buffer containing various concentrations of CaCl2 (0.5 mM unless otherwise stated).

Statistical Analysis—Data are presented as mean ± S.E. and statistical significance was determined by one-way analysis of variance (Tukey post hoc test) or by unpaired t test as appropriate.


Figure 2
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  		FIGURE 2.
CaRpT888 immunoreactivity in cells expressing wild-type or mutant CaR. HEK cells stably transfected with either wild-type CaR (wt) or CaRT888A mutant (T888A) receptors were PMA-treated and processed either for immunoblotting (A) or immunofluorescence (B) using either CaRpT888-specific antibody (A and B, upper panels) or total CaR antibody (B, lower panels) as described in the legend to Fig. 1. Results shown are representative of a minimum of three independent experiments.

 


Figure 3
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  		FIGURE 3.
Agonist-induced CaRT888 phosphorylation. A, CaR-HEK cells were preincubated in the presence or absence of 250 nM GF109203X (5 min), then exposed to Experimental Buffer supplemented with either an additional 2 mM Ca2+o (2.5 mM final), 1 µM NPS R-467, GF109203X, or a combination of treatments as indicated, for a further 10 min. Immunoblotting was then performed on the resulting cell lysates using anti-CaRpT888 antibody and quantified by densitometry. Data are shown histographically (n = 4) below a representative blot. *, p < 0.05 versus control; +++, p < 0.001 versus 2.5 Ca2+o/NPS R-467. B, cells treated as in A were processed for immunofluorescence. Results are representative of a minimum of three independent experiments.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
CaR-HEK cells were treated with the phorbol ester, phorbol 12-myristate 13-acetate (PMA) to stimulate PKC activity and the resulting change in CaRT888 phosphorylation was investigated by immunoblotting and immunofluorescence. The affinity purified, phospho-specific anti-CaRpT888 antibody detected a PMA-induced increase in immunoreactivity in two protein bands (Fig. 1A, panel i) corresponding to the high-mannose "core"-glycosylated CaR (140 kDa) and the mature glycosylated CaR (160 kDa) (12). To confirm the specificity of the signal, the following experiments were performed. First, to demonstrate that the protein bound to the anti-CaRpT888 antibody possesses the same biochemical properties as CaR, the PMA-pretreated CaR-HEK cell lysates were solubilized in Laemmli buffer in the presence or absence of reducing agent. Because native CaR is a disulfide-linked dimer (13, 14), it exhibits dimer-like electrophoretic mobility in the absence of reducing agent but is solubilized to monomers in its presence. Here, both anti-CaRpT888 polyclonal and anti-CaR monoclonal (ADD, i.e. total CaR) antibodies detected CaR-like monomers where the lysates were solubilized with beta-mercaptoethanol and dimeric immunoreactivity under non-reducing conditions (Fig. 1 A, panel ii). Second, the anti-CaR monoclonal ADD antibody immunodetected CaR-sized proteins in immunoprecipitated pellets generated using the anti-CaRpT888 antibody (Fig. 1A, panel iii). CaR-reactive signal was observed in the samples pretreated with PMA in the absence or presence of calyculin A (PP1/PP2A protein phosphatase inhibitor), but was absent in unstimulated cells. Next, to confirm that the CaRpT888 antibody was indeed phospho-specific, the PMA-induced immunoreactivity detected in CaR-HEK cells was ablated by pretreating the anti-CaRpT888 antibody with phosphorylated immunizing peptide but not when using non-phosphorylated peptide at an identical concentration (Fig. 1B, panel i). Similar results were obtained when cells grown on glass coverslips where incubated in the presence or absence of PMA and then fixed and stained with anti-CaRpT888 antibody. PMA treatment increased the resulting immunofluorescence but this staining was ablated by the phosphorylated peptide (Fig. 1B, panel ii). At very high peptide concentrations (≥1 µM), both phosphorylated and non-phosphorylated peptides ablated antibody binding, whereas at very low peptide concentrations (<1 nM) neither ablated the signal (data not shown). Finally, CaR-like, phorbol-sensitive immunoreactivity was observed in the lysates of HEK cells stably transfected with wild-type CaR but not with a mutant receptor in which Thr888 was replaced with alanine (CaRT888A; Fig. 2A). Similar observations were made by immunofluorescence in which PMA elicited anti-CaRpT888 immunoreactivity in CaR-HEK cells but not in CaRT888A-HEK cells despite both cell types exhibiting equivalent immunoreactivity to the ADD monoclonal antibody (Fig. 2B).

To examine whether CaRT888 phosphorylation is altered by receptor stimulation, CaR-HEK cells were incubated for 10 min in buffer containing increasing Ca2+o concentrations in the presence or absence of the calcimimetic NPS R-467 (CaR positive allosteric modulator). Increasing Ca2+o concentration from 0.5 to 2.5 mM induced a small rise in CaRT888 phosphorylation (Fig. 3A), whereas addition of NPS 467-R (1 µM) increased it further (Fig. 3, A and B; +42 ± 10%, p < 0.05). Cotreatment with the PKC inhibitor GF109203X inhibited the signal substantially (-60 ± 3%, p < 0.001). These responses were qualitatively similar to the immunofluorescence observed in situ in CaR-HEK cells grown on coverslips and stained as before. Under control conditions (0.5 mM Ca2+o-containing buffer), NPS R-467 and GF109203X were without effect (data not shown). None of the above responses were observed in HEK cells stably transfected with empty vector (data not shown).


Figure 4
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  		FIGURE 4.
Effects of calcimimetic and calcilytic treatment on CaRT888 phosphorylation. CaR-HEK cells were preincubated in the presence or absence of 1 µM NPS-89636 (5 min), then exposed for a further 10 min to Experimental Buffer containing either 2.5 or 5 mM Ca2+o, 1 µM NPS R-467 or S-467 or NPS-89636 as labeled. Cells were then fixed and processed for immunofluorescence using anti-CaRpT888 antibody. Results are representative of three independent experiments.

 


Figure 5
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  		FIGURE 5.
Extracellular Ca2+-induced Ca2+i oscillations in wild-type and T888A mutant CaRs. A, Fura-2-loaded CaR-HEK (i) or CaRT888A-HEK (ii) cells were treated with increasing concentrations of Ca2+o (0.5-10 mM) and the resulting changes in Ca2+i concentration measured by single cell microfluorometry. Each panel shows traces from four representative cells with a fifth trace at the bottom showing the "global" response from at least 30 cells per coverslip. Results are representative of a minimum of six independent experiments. B, the pattern of Ca2+i response in each cell (minimum of 120) was categorized either as non-responding (including cells exhibiting a small, slow rise in Ca2+i because of non-receptor mediated Ca2+ influx), transient, oscillatory, or sustained. Data shown include the responses to 2 and 3 mM Ca2+o in cells stably expressing wild-type (wt) CaR or CaRT888A (T888A). C, concentration-effect curve for Ca2+o-induced Ca2+i mobilization obtained from the experiments detailed in A. CaRT888A exhibited a leftward shifted concentration-effect curve relative to wild-type CaR, with a significantly reduced EC50 value (unpaired t test; see inset).

 
The effect of NPS R-467 on CaRT888 phosphorylation was stereoselective as 1 µM NPS S-467 was without effect (Fig. 4). Replacement of NPS R-467 with an additional 2.5 mM CaCl2 (i.e. 5 mM final Ca2+o concentration) also elicited additional CaRT888 phosphorylation and this effect was abolished by cotreatment with the calcilytic NPS-89636 (1 µM; CaR negative allosteric modulator). High Ca2+o-induced CaRT888 phosphorylation was also observed in cells treated at room temperature (data not shown).

Previous studies have shown that the mutant receptor CaRT888A exhibits greater sensitivity to Ca2+o than the wild-type receptor (8) and that stimulation of CaRT888A with 3 mM Ca2+o elicits non-oscillatory, sustained Ca2+i mobilization compared with the oscillatory behavior observed with the wild-type receptor (9). To examine this further, we compared the responses of wild-type CaR and CaRT888A to various concentrations of Ca2+o using single-cell epifluoresence Ca2+i imaging (Fig. 5). In wild-type CaR-HEK cells, increasing Ca2+o concentration from 0.5 to 2 mM induced a transient response in some cells (16%), whereas most of the others were non-responsive (Fig. 5, A, panel i, and B). However, in cells stably transfected with CaRT888A, 2 mM Ca2+o elicited responses in 58% of cells including slow oscillations and sustained responses (Fig. 5, A, panel ii, and B). Following subsequent treatment with 3 mM Ca2+o, virtually all of the CaR-HEK cells responded, with the majority exhibiting oscillations although with 32% showing sustained Ca2+i mobilization. In the CaRT888A-HEK cells, the majority of responsive cells exhibited sustained responses although 25% still exhibited slow oscillations. To compare the Ca2+o sensitivity of CaRT888A versus wild-type receptor, the dose-response data for each cell were pooled, revealing that the EC50 of 3.4 mM for wild-type CaR was reduced to 2.6 mM Ca2+o in the CaRT888A cells, consistent with a previous study in which the receptors were expressed transiently (8). CaR-induced Ca2+i oscillations initiated by 100 µM neomycin were maintained for up to 5 min in both Ca2+-containing (0.5 mM) and Ca2+-free (supplemented with 1 mM EGTA) buffer but were abolished under Ca2+-free conditions by thapsigargin pretreatment demonstrating the involvement of intracellular Ca2+ mobilization in the responses (data not shown).

Previous studies have shown that phorbol ester treatment decreases the Ca2+o sensitivity of the CaR, whereas PKC inhibition increases it (7-9), however, the effect of down-regulating PKC expression via chronic phorbol ester pretreatment has not been reported in this context. Here we found that in wild-type CaR-HEK cells, chronic PMA pretreatment appears to increase the sensitivity of the receptor and elicits non-oscillatory, sustained responses in most cells (Fig. 6). In CaRT888A cells, chronic PMA pretreatment had little overall effect on Ca2+o sensitivity but resulted in responses that were mostly sustained.


Figure 6
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  		FIGURE 6.
Effect of chronic phorbol ester pretreatment on CaR-induced intracellular Ca2+ responses. A, intracellular Ca2+ levels were measured in wtCaR-HEK and CaRT888A-HEK cells as described in the legend to Fig. 5. Cells were first pretreated for 16 h with PMA (1µM) to down-regulate phorbol-sensitive PKC isoforms. Cell responses (minimum of 180) were categorized as before (B) and the concentration-effect curve (C) was used to estimate the EC50 values of the responses. n ≥ 8.

 
Next we examined the effect on CaR-induced Ca2+i oscillations of a non-selective PKC inhibitor (GF109203X) compared with the effects of two conventional PKC (i.e. Ca2+-dependent)-selective inhibitors namely Gö6976 and an anilinomonoindolylmaleimide beta-selective PKC inhibitor (15). Addition of GF109203X to cells exhibiting Ca2+i oscillations produces sustained responses in most (≥80%) of the cells (Fig. 7). Where cells exhibiting oscillations were first treated with either 300 nM Gö6976 or 500 nM PKCbeta inhibitor, conversion from oscillatory to sustained Ca2+i mobilization was observed in 40.1 and 27.5%, respectively, of the cells sensitive to subsequent GF109203X treatment. This concentration of PKCbeta inhibitor should also inhibit PKC{alpha} and PKC{gamma}, whereas no change in the Ca2+i oscillations were seen in response to 50 nM PKCbeta inhibitor that should be selective for that isozyme (15).

Next, to identify a candidate phosphatase for the dephosphorylation of CaRT888, CaR-HEK cells were pre-treated acutely with PMA (10 min) and then incubated for a further 5 min in the presence or absence of various phosphatase inhibitors. In each case, GF109203X (250 nM) was included during the 5-min incubation to prevent any further phosphorylation. In the absence of any phosphatase inhibitors, substantial dephosphorylation of CaRT888 was observed within 5 min, however, in the presence of the PP1/PP2A inhibitor calyculin A (100 nM), dephosphorylation was prevented (Fig. 8A). In contrast, tautomycin (PP1 inhibitor) and FK506 (PP2B inhibitor) had little effect on CaRT888 dephosphorylation (not shown). To examine this further, the colocalization of PP2A and CaRpT888 was investigated by dual-fluorophore immunofluorescence. CaR-HEK cells were incubated acutely in PMA and then fixed and stained with the phospho-specific anti-CaRpT888 antibody and a monoclonal anti-PP2A catalytic subunit antibody, followed by goat anti-rabbit (Alexa 488) and donkey anti-mouse (Alexa 594) antibodies, respectively. Merging of the resulting green (CaRpT888) and red (PP2A) channels reveals partial colocalization (yellow) of the 2 antigens (Fig. 8B). Finally, to determine whether phosphatase inhibition affects CaR function, Fura-2-loaded CaR-HEK cells were exposed to 2.5 mM Ca2+o to induce Ca2+i oscillations and then switched to an identical buffer supplemented with 100 nM calyculin, 500 µM endothall thioanhydride, or 5 nM tautomycin. Calyculin and endothall both inhibited the Ca2+i oscillations reversibly, whereas tautomycin was without effect.

To examine whether the effects of PMA and calyculin on CaR function are specific to Thr888, CaRT888A-HEK cells were stimulated with 2 mM Ca2+o to elicit either oscillatory or sustained Ca2+i mobilization and then cotreated with either drug. Despite the absence of Thr888, PMA still suppressed receptor-mediated Ca2+i mobilization (Fig. 9A). Increasing the Ca2+o concentration to 3 and then to 5 mM overcame the inhibitory effect of PMA. Interestingly, the PMA tended to ablate the Ca2+i oscillations while having little effect on the rate of decline of the "sustained" responses. Similarly, cotreatment with calyculin ablated CaRT888A-induced Ca2+i oscillations but had little effect on cells exhibiting a sustained response (Fig. 9B). Following the removal of calyculin, 5 mM Ca2+o elicited a potent response in all cells demonstrating their continued viability.

Finally, bovine parathyroid cells were incubated in the absence or presence of PMA/calyculin, fixed, and then examined for their CaRpT888 content as before. The effect of PMA/calyculin cotreatment was to increase endogenous CaRT888 phosphorylation in the parathyroid cells (Fig. 10A). In addition, in Fura-2-loaded bovine parathyroid cells, the rise in [Ca2+]i elicited by increasing [Ca2+]o from 0.5 to 2 mM, was suppressed by cotreatment with PMA (Fig. 10B).


Figure 7
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  		FIGURE 7.
Effect of PKC inhibition on CaR-induced intracellular Ca2+ responses. A, intracellular Ca2+ levels were measured in wtCaR-HEK as described in the legend to Fig. 5. Ca2+i oscillations (induced by 2.5 mM Ca2+o) were unaffected by sham changes in solution (indicated by the downward arrow) but were converted to sustained responses by GF109203X (250 nM). B, cotreatment with the conventional PKC-selective inhibitor Gö6976 (300 nM) converted oscillations to sustained responses in 40% of the cells (as represented by Cell 1) but not in the remainder (Cell 2). C, cotreatment with the PKCbeta-selective inhibitor (500 nM) converted oscillations to sustained responses in 28% of the cells (as represented by Cell 1) but not in the remainder (Cell 2). Data are from a minimum of 90 cells from three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Given the key role of the CaR in controlling extracellular calcium homeostasis, the regulation of its function has important consequences for whole animal physiology (1, 2). A role for PKC-mediated phosphorylation in the regulation of CaR activity has been proposed before, based on the use of pharmacologic modulators of PKC activity and the expression of mutant receptors lacking certain PKC consensus sites (3-5, 7-9). However, to prove conclusively that CaRT888 can be phosphorylated in vivo and then to determine the agonist sensitivity of such phosphorylation, as well as to identify a candidate CaR phosphatase, we raised a phospho-specific polyclonal antibody to residue Thr888 of the human CaR (6).

The CaRpT888-reactive protein bands visible by immunoblotting correspond in size to the high mannose and fully mature forms of CaR (12) and behave as disulfide-linked dimers (13, 14) in the absence of reducing agent. In addition, the monoclonal antibody (ADD) recognizing total CaR detected the same bands as above in immunoprecipitates pulled-down using the phosphospecific antibody, whereas no anti-CaRpT888 immunoreactivity was observed in empty vector-transfected HEK cells. Together, these data demonstrate that the anti-CaRpT888 antibody detects CaR as opposed to an endogenous HEK cell protein.


Figure 8
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  		FIGURE 8.
Effect of protein phosphatase inhibition on CaRT888 dephosphorylation and CaR-induced Ca2+i oscillations. A, wtCaR-HEK cells were incubated in the absence (lane 1) or presence (lanes 2-4) of PMA (1 µM, 10 min) to increase basal CaRT888 phosphorylation and either lysed immediately (lanes 1 and 2) or incubated for a further 5 min (lanes 3 and 4) in the presence of GF109203X (250 nM), further supplemented with (lane 4) or without (lane 3) the PP1/PP2A inhibitor calyculin A (100 nM). These cells were then lysed and processed with the earlier lysates for immunoblotting against the anti-CaRpT888 antibody (n ≥ 4). B, PMA-stimulated CaR-HEK cells were fixed and costained with the polyclonal anti-CaRpT888 antibody and a monoclonal anti-PP2A catalytic subunit antibody, followed by goat anti-rabbit (Alexa 488) and donkey anti-mouse (Alexa 594) antibodies. Merging of the resulting green (CaRpT888) and red (PP2A) signal reveals partial colocalization (yellow) of the 2 antigens. C, Fura-2-loaded CaR-HEK cells were exposed to 2.5 mM Ca2+o to induce Ca2+i oscillations and then switched to an identical buffer supplemented with 100 nM calyculin A, 500 µM to 1 mM endothall, or 5 nM tautomycin. Cells were then washed in Experimental Buffer and stimulated with 3 mM Ca2+o-containing buffer to demonstrate continued viability of the responses. Data representative of at least 90 cells from a minimum of three independent experiments are shown.

 
Next, ablation of the signal with the phosphorylated immunizing peptide but not the non-phosphorylated peptide demonstrates the phosphospecificity of the CaRpT888 antibody. This is supported by the observation that cells stably expressing CaRT888A exhibited no immunoreactivity with anti-CaRpT888 but normal immunoreactivity with the ADD monoclonal antibody.

Having validated the antibody and demonstrated phorbol-sensitive CaRT888 phosphorylation, it was then possible to demonstrate that acute calcimimetic treatment elicited CaRT888 phosphorylation in a stereoselective manner. This response was mimicked by an additional increase in Ca2+o concentration, an effect that was inhibited by cotreatment with the calcilytic NPS-89636 (11, 16, 17). Therefore, CaR activation elicits feedback phosphorylation of its own intracellular domain residue Thr888. As CaRT888 phosphorylation could be induced by acute PMA treatment and inhibited by GF109203X it is highly likely that the phosphorylation is PKC-dependent.


Figure 9
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  		FIGURE 9.
Effect of phorbol ester and calyculin treatment on CaRT888A-induced intracellular Ca2+ responses. A, intracellular Ca2+ levels were measured in CaRT888A-HEK cells in buffer containing 0.5 mM Ca2+o (unless otherwise stated) as described in the legend to Fig. 5. Increasing Ca2+o concentrations to 2 mM elicited oscillatory Ca2+i mobilization in some cells (e.g. Cell 1) and sustained responses in others (e.g. Cell 2). After 6 min, PMA (1 µM) was added and later the Ca2+o concentration was increased further (to 3 and then 5 mM) in the continued presence of PMA. Similar responses were seen with 8 separate coverslips (≥40 cells). B, CaRT888A-HEK cells were stimulated with 2 mM Ca2+o to induce oscillatory (Cell 1) or sustained (Cell 2) responses as before, prior to the addition of 100 nM calyculin for 4 min. Cells were allowed to recover for 3 min in 2 mM Ca2+o and then treated with 5 mM Ca2+o to demonstrate that the cells were still responsive. Similar responses were observed with three separate coverslips (≥25 cells).

 
CaRpT888 immunoreactivity was observed in a 160-kDa band, most likely the fully mature receptor, and a 140-kDa band, presumed to be the high mannose, core-glycosylated protein. Whereas feedback phosphorylation of the mature, membrane-localized receptor could regulate receptor function or Ca2+i oscillation generation/maintenance, it is less clear what purpose phosphorylating the immature receptor would serve. For example, it could regulate the maturation or forward trafficking of the CaR to the membrane. It will be interesting to determine whether under certain conditions, phosphorylation of the two bands can occur differentially, for example, by different PKC isozymes or with non-identical time courses. Thus, the current study establishes that PKC-mediated phosphorylation of the mature CaR can occur, although possibly in addition to phosphorylation of intracellular or immature receptors.

In contrast to a previous report (9), we did not find that CaRT888A produced exclusively sustained Ca2+i responses. Indeed, in 2 mM Ca2+o, more cells exhibited oscillations than sustained responses, although in 3 mM Ca2+o most cells did exhibit sustained responses similar to the data reported previously (9). Nevertheless, even in 3 mM Ca2+o, some CaRT888A-HEK cells still exhibited oscillations and this should not be possible if dynamic changes in CaRT888 phosphorylation account exclusively for Ca2+i oscillations. It should be noted, however, that the oscillations induced by CaRT888A here are much slower than those elicited by the wild-type CaR. The reason for the apparent discrepancy between the two studies is unclear. In the previous study (9), transient transfection was employed, whereas here we studied HEK cells stably expressing CaRT888A. Given the heightened sensitivity of CaRT888A it is possible that in generating the stable clone, the most responsive cells died and thus slightly less responsive cells were selected. In support of this, we noted during the selection process that the culture of CaRT888A-HEK cells in Dulbecco's modified Eagle's medium (containing 1.8 mM CaCl2) resulted in substantial cell death necessitating the use of RPMI media instead (data not shown). In CaRT888A-HEK cells stimulated with 2 mM Ca2+o, the addition of either PMA or calyculin had little effect on the sustained responses, but instead inhibited Ca2+i oscillations. Increasing the Ca2+o concentration, in the continued presence of PMA, overcame the inhibitory effect of the phorbol ester. Because PMA and calyculin act to increase or sustain Ser/Thr phosphorylation, respectively, these data could be explained by a PKC-mediated decrease in CaR sensitivity that occurs despite the absence of Thr888. If true, these data could suggest that there is a further signaling determinant within CaR or its associated signaling machinery for the establishment and maintenance of Ca2+i oscillations. In support of this is the fact that chronic PMA pretreatment virtually abolished Ca2+i oscillations in CaRT888A-HEK cells (>98%) and did completely abolish oscillations in wild-type CaR-HEK cells. In the current study, chronic PMA pretreatment elicited heightened Ca2+o sensitivity in wild-type CaR-HEK cells in a manner similar to the effects of PKC inhibitors published previously (7, 9). Chronic PMA pretreatment most likely down-regulates conventional and novel PKC isoforms (18) and PKC{alpha} and -{epsilon} have been reported to be activated upon CaR stimulation in parathyroid and CaR-HEK cells (19). These data are consistent with the previous observation (20) that CaR-induced inositol 1,4,5-trisphosphate formation was heightened in bovine parathyroid cells pre-exposed overnight to PMA (1 µM). However, in that study the Ca2+o-induced Ca2+i mobilization dose-response curve was not leftward shifted and neither was the suppressive effect of 2 mM Ca2+o on PTH secretion significantly enhanced, although its inhibitory effect may have already been close to maximal under those conditions (20). PKC inhibitors selective for the conventional PKC isozymes partially mimicked the effect of the non-selective PKC inhibitor GF109203X in converting oscillatory Ca2+i responses into sustained responses, albeit in no more than 40% of the cells. Thus, together with the data from the chronic PMA pretreatment experiments, these data could suggest that a combination of conventional and novel PKC isozymes contribute to CaR regulation.


Figure 10
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  		FIGURE 10.
Effects of calyculin and phorbol treatment on CaRT888 phosphorylation and intracellular Ca2+i responses in bovine parathyroid cells. A, bovine parathyroid cells adherent on glass coverslips were incubated at 37 °C for 5 min in the absence or presence of PMA (1 µM)/calyculin (100 nM), fixed, and then stained with CaRpT888 antibody as before (panel i). CaRpT888 cellular immunoreactivity was quantified and is shown histographically in panel ii (n = 12 from a minimum of three independent experiments; *, p < 0.05 by unpaired t test). B, in Fura-2-loaded bovine parathyroid cells, the rise in [Ca2+]i elicited by increasing [Ca2+]o from 0.5 to 2 mM, was suppressed by cotreatment with PMA (1 µM). In the experiment shown, the CaR responsiveness of the cells was first established using the calcimimetic NPS R-467 (1 µM). The upper panel shows a representative trace from a single cell, whereas the lower panel shows the combined response of a cluster of 6 cells (global). These results are representative of the responses obtained on at least six coverslips from three independent experiments.

 
Spontaneous dephosphorylation of CaRT888 occurred in the 5 min following a 10-min PMA pretreatment indicating the presence of an active phosphatase at the CaR intracellular domain. As this effect was largely unaltered by selective inhibitors of the phosphatases PP1 and PP2B, but ablated by the PP1/PP2A inhibitor calyculin (21) and the PP2A-selective inhibitor endothall, this suggests that PP2A, or at least a calyculin-sensitive phosphatase, is responsible for this dephosphorylation. Indeed, calyculin decreased CaR-induced oscillation frequency and in some cells suppressed them entirely, consistent with the idea that CaRT888 phosphorylation is inhibitory to Ca2+i mobilization. Larger concentrations of calyculin gave much greater inhibitory effects (not shown). However, these cells also tended to detach rapidly from the coverslip as described for other cell types (22, 23). Thus, only experiments in which normal oscillations were recoverable following the removal of calyculin are presented. The inhibitory effect of calyculin was mimicked by 500 µM endothall thioanhydride, a protein phosphatase inhibitor (24) that exhibits greater selectivity for PP2A over PP1 (IC50, PP2A, 90 nM; PP1, 5 µM). In contrast, the PP1-selective inhibitor tautomycin (5 nM) was without effect (IC50, PP1, 1 nM; PP2A 10 nM). In further support of the involvement of PP2A in CaRT888 dephosphorylation, the catalytic domain of PP2A colocalized, partially, with CaRpT888. Together, these data implicate a calyculin-sensitive phosphatase, most likely PP2A, as the phosphatase responsible for CaRT888 dephosphorylation. Thus it will be interesting to investigate CaR signaling in cells underexpressing or overexpressing the various protein phosphatases.

Finally, anti-CaRpT888 immunoreactivity was also observed in bovine parathyroid cells treated with PMA in the presence of calyculin indicating the potential utility of this antibody for studying CaR phosphorylation in endogenous expression systems. For example, the antibody could be used to examine whether the loss of parathyroid Ca2+o sensitivity seen in conditions such as renal disease or aging involves CaR phosphorylation as opposed to down-regulation. Also, it could be used to test whether circadian changes in PTH secretion might be explained by changes in CaRT888 phosphorylation and thus Ca2+o sensitivity, over the course of the day.

Together these data confirm residue Thr888 plays a predominant role in the regulation of Ca2+i oscillations but that a further PKC-dependent mechanism could be involved, perhaps even a second phosphorylation site. In addition, we demonstrate for the first time that CaR activation induces feedback phosphorylation of the intracellular domain residue Thr888 and that this residue is a substrate for a calyculin-sensitive protein phosphatase.


   FOOTNOTES
 
* This work was supported in part by Biotechnology and Biological Sciences Research Council Grant BBSB04986. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Floor 2, Core Technology Facility, University of Manchester, 46 Grafton St., Manchester M13 9NT, United Kingdom. Tel.: 44-161-275-5459; Fax: 44-161-275-5600; E-mail: d.ward{at}manchester.ac.uk.

2 The abbreviations used are: CaR, calcium-sensing receptor; PTH, parathyroid hormone; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PP2, protein phosphatase 2. Back


   ACKNOWLEDGMENTS
 
We thank Prof. Arthur Conigrave (University of Sydney, Australia) for providing the CaRT888A mutant and Dr. Ed Nemeth (NPS Pharmaceuticals, Inc., Toronto, Canada) for supplying the calcimimetic and calcilytic reagents. We further thank them, as well as Dr. Daniela Riccardi (University of Cardiff, UK), Dr. Austin Elliott (University of Manchester), Claire Gibbons (University of Manchester, UK), and Peter March (Bioimaging Facility, Faculty of Life Sciences, University of Manchester, UK) for advice and technical assistance. Finally, we thank Dr. Richard Murray (University of Liverpool, UK) for help in obtaining bovine parathyroid glands.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Brown, E. M., Gamba, G., Riccardi, D., Lombardi, M., Butters, R., Kifor, O., Sun, A., Hediger, M. A., Lytton, J., and Hebert, S. C. (1993) Nature 366, 575-580[CrossRef][Medline] [Order article via Infotrieve]
  2. Brown, E. M., and MacLeod, R. J. (2001) Physiol. Rev. 81, 239-297[Abstract/Free Full Text]
  3. Membreno, L., Chen, T. H., Woodley, S., Gagucas, R., and Shoback, D. (1989) Endocrinology 124, 789-797[Abstract]
  4. Racke, F. K., and Nemeth, E. F. (1993) J. Physiol. 468, 141-162[Abstract/Free Full Text]
  5. Racke, F. K., and Nemeth, E. F. (1993) J. Physiol. 468, 163-176[Abstract/Free Full Text]
  6. Garrett, J. E., Capuano, I. V., Hammerland, L. G., Hung, B. C., Brown, E. M., Hebert, S. C., Nemeth, E. F., and Fuller, F. (1995) J. Biol. Chem. 270, 12919-12925[Abstract/Free Full Text]
  7. Bai, M., Trivedi, S., Lane, C. R., Yang, Y., Quinn, S. J., and Brown, E. M. (1998) J. Biol. Chem. 273, 21267-21275[Abstract/Free Full Text]
  8. Jiang, Y. F., Zhang, Z., Kifor, O., Lane, C. R., Quinn, S. J., and Bai, M. (2002) J. Biol. Chem. 277, 50543-50549[Abstract/Free Full Text]
  9. Young, S. H., Wu, S. V., and Rozengurt, E. (2002) J. Biol. Chem. 277, 46871-46876[Abstract/Free Full Text]
  10. Ward, D. T., McLarnon, S. J., and Riccardi, D. (2002) J. Am. Soc. Nephrol. 13, 1481-1489[Abstract/Free Full Text]
  11. Davies, S. L., Gibbons, C. E., Vizard, T., and Ward, D. T. (2006) Am. J. Physiol. 290, C1543-C1551[CrossRef]
  12. Bai, M., Quinn, S., Trivedi, S., Kifor, O., Pearce, S. H., Pollak, M. R., Krapcho, K., Hebert, S. C., and Brown, E. M. (1996) J. Biol. Chem. 271, 19537-19545[Abstract/Free Full Text]
  13. Ward, D. T., Brown, E. M., and Harris, H. W. (1998) J. Biol. Chem. 273, 14476-14483[Abstract/Free Full Text]
  14. Bai, M., Trivedi, S., and Brown, E. M. (1998) J. Biol. Chem. 273, 23605-23610[Abstract/Free Full Text]
  15. Tanaka, M., Sagawa, S., Hoshi, J., Shimoma, F., Matsuda, I., Sakoda, K., Sasase, T., Shindo, M., and Inaba, T. (2004) Bioorg. Med. Chem. Lett. 14, 5171-5174[CrossRef][Medline] [Order article via Infotrieve]
  16. Nemeth, E. F., Delmar, E. G., Heaton, W. L., Miller, M. A., Lambert, L. D., Conklin, R. L., Gowen, M., Gleason, J. G., Bhatnagar, P. K., and Fox, J. (2001) J. Pharmacol. Exp. Ther. 299, 323-331[Abstract/Free Full Text]
  17. Dvorak, M. M., Siddiqua, A., Ward, D. T., Carter, D. H., Dallas, S. L., Nemeth, E. F., and Riccardi, D. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 5140-5145[Abstract/Free Full Text]
  18. Nishizuka, Y. (1986) Science 233, 305-312[Abstract/Free Full Text]
  19. Sakwe, A. M., Larsson, M., and Rask, L. (2004) Exp. Cell Res. 297, 560-573[CrossRef][Medline] [Order article via Infotrieve]
  20. Racke, F. K., and Nemeth, E. F. (1994) Am. J. Physiol. 267, E429-E438[Medline] [Order article via Infotrieve]
  21. Ishihara, H., Martin, B. L., Brautigan, D. L., Karaki, H., Ozaki, H., Kato, Y., Fusetani, N., Watabe, S., Hashimoto, K., Uemura, D., and Hartshorne, D. J. (1989) Biochem. Biophys. Res. Commun. 159, 871-877[CrossRef][Medline] [Order article via Infotrieve]
  22. Hosoya, N., Mitsui, M., Yazama, F., Ishihara, H., Ozaki, H., Karaki, H., Hartshorne, D. J., and Mohri, H. (1993) J. Cell Sci. 105, 883-890[Abstract]
  23. Chartier, L., Rankin, L. L., Allen, R. E., Kato, Y., Fusetani, N., Karaki, H., Watabe, S., and Hartshorne, D. J. (1991) Cell Motil. Cytoskeleton. 18, 26-40[CrossRef][Medline] [Order article via Infotrieve]
  24. Li, Y. M., Mackintosh, C., and Casida, J. E. (1993) Biochem. Pharmacol. 46, 1435-1443[CrossRef][Medline] [Order article via Infotrieve]

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R. Mamillapalli, J. VanHouten, W. Zawalich, and J. Wysolmerski
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