Protein Kinase C Phosphorylation of Threonine at Position 888 in Ca2+ o -Sensing Receptor (CaR) Inhibits Coupling to Ca2+ Store Release*

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+ i dynamics 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 (Ca 2؉ o )-evoked stimulation of phospholipase C and the associated increases in cytosolic calcium (Ca 2؉ 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 Ca 2؉ 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 Ca 2؉
i due to release of Ca 2؉ from intracellular stores. Consistent with the effect being mediated by activation of PKC, the inhibitory effect of PKC activators on Ca 2؉ 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 Ca 2؉ mobilization. The effect of PKC activation can be maximally blocked by mutating three PKC sites (Thr 888 , Ser 895 , and Ser 915 ) or all five PKC sites. In vitro phosphorylation shows that Thr 888 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 Ca 2؉ oevoked changes in Ca 2؉ i dynamics in parathyroid cells.

The extracellular calcium concentration (Ca 2ϩ
o ) is tightly regulated by the interactions of several hormones (e.g. parathyroid hormone (PTH), 1 vitamin D, and calcitonin) and organ systems (i.e. parathyroid gland, kidney, bone, and intestine) (1)

. Parathyroid cells respond to changes in Ca 2ϩ
o with oppo-sitely directed alterations in PTH secretion through a cell surface, G protein-coupled receptor, the Ca 2ϩ o -sensing receptor (CaR).
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. Ca 2ϩ o and Mg 2ϩ o ), trivalent cations (e.g.

Gd 3ϩ
o ) and polyamines (e.g. neomycin) (2). In response to increases in Ca 2ϩ o , the CaR stimulates accumulation of inositol phosphates and produces transient followed by sustained increases in Ca 2ϩ i . Subsequently, cDNAs encoding the human homologue of the same receptor have been cloned from parathyroid (3) and kidney (4) 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 (36,37).
High Ca 2ϩ o -evoked suppression of PTH secretion and the concurrent increases in Ca 2ϩ i in parathyroid cells can be negatively regulated by activation of protein kinase C (PKC) (5)(6)(7)(8)(9)(10)(11)(12)(13) . Such negative regulation by PKC has been suggested to be involved in the reduced responsiveness of adenomatous or hyperplastic parathyroid glands to Ca 2ϩ o as a result of an increase in membrane-associated PKC (14,15), although there is also reduced expression of the CaR in these glands (16,17). Likewise, PKC may contribute to age-related changes in the regulation of PTH secretion by Ca 2ϩ o in rats (18). Therefore, stimulus-secretion coupling in parathyroid cells can be modulated by PKC, perhaps at an early step in the process of Ca 2ϩ 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 Ca 2ϩ o by covalently modifying these sites. To test this hypothesis, we have transiently transfected a human parathyroid CaR cDNA (18) in HEK293 cells and mutated each of the five putative PKC sites individually or in varying combinations in the CaR. We studied Ca 2ϩ i responses of the wild type and mutant CaRs to elevations in Ca 2ϩ o and the polycationic CaR agonist, neomycin, in the presence or absence of various PKC activators (e.g. phorbol myristate acetate (PMA), Mezerein, and (Ϫ)-Indolactam V) and/or the PKC inhibitor, staurosporine. Our results show that phosphorylation by PKC at one of the five predicted PKC phosphorylation sites (Thr 888 ) substantially reduces CaR-mediated release of Ca 2ϩ from intracellular stores. Therefore, it is possible that PKC phosphorylation of the CaR regulates PTH secretion by inhibiting Ca 2ϩ mobilization or perhaps generation of some other intracellular mediator(s) along the inositol trisphosphate/phospholipase C pathway.

EXPERIMENTAL PROCEDURES
Site-directed Mutagenesis-Site-directed mutagenesis was performed using the approach described by Kunkel (19) 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 (20). 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. DH5␣ 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 (20). Likewise, mutated versions of cassette 6 containing the desired mutations were doubly digested with XhoI and XbaI and cloned into the reconstructed receptor in pcDNA3.
Construction of a Mutant CaR with Mutations of Two PKC Sites (S895A/S915A)-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 and XbaI. The resultant clone was confirmed by sequencing.
Construction of a Mutant CaR with Mutations of Two PKC Sites (T646V/S794A)-The mutant receptor carrying T646V was doubly digested with HpaI and XhoI, and the mutant receptor carrying S794A was doubly digested with XhoI and XbaI. 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 and XbaI. The resultant clone was confirmed by sequencing to carry these two mutations.
Construction of a Mutant CaR with Five Mutated PKC Sites-Cassette 6 carrying S794A was doubly digested with XhoI and SphI, and the same cassette carrying three mutations was doubly digested with SphI and XbaI. 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.
Construction of a Mutant CaR with Four Mutated PKC Sites with Thr 888 unchanged (T646V/S794A/S895A/S915A)-The receptors carrying five mutated PKC sites and two mutated sites (S895A/S915A) was doubly digested with KpnI and XbaI 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.
Construction of Flag-tagged CaRs-The Flag, an epitope tag, was introduced into the third cassette of the wild type CaR as described previously (21). 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.
Transient Expression of CaRs in HEK293-CaR cDNAs were prepared using the Midi Plasmid Kit (Qiagen). LipofectAMINE (Life Technologies, Inc.) was employed as a DNA carrier for transfection (22). 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 Lipo-fectAMINE 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 re-duced serum medium and added to 90% confluent HEK293 cells plated on 13.5 ϫ 20.1-mm glass coverslips using 0.625 g of DNA (for measurement of Ca 2ϩ i ) or in 100 mm Petri dishes using 3.75 g 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 Ca 2ϩ o -sensing receptor protein was assayed 48 h after the start of transfection.
Measurement of Ca 2ϩ i by Fluorometry in Cell Populations-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 mM HEPES, pH 7.4, containing 125 mM NaCl, 4 mM KCl, 1.25 mM CaCl 2 , 1 mM MgSO 4 , 1 mM NaH 2 PO 4 , 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 mM HEPES, pH 7.4, containing 125 mM NaCl, 4 mM KCl, 0.5 mM CaCl 2 , 0.5 mM MgCl 2 , 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 (23). 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 Ϯ 40 nm through a wide-band emission filter. The ratio of emitted light (340/380 excitation) was used as readout for Ca 2ϩ i as described previously (23). For PKC activation, the cells were preincubated with the PKC activators (PMA, mezerein, or (Ϫ)-indolactam V) for 1-2 min. For PKC inhibition, the cells were preincubated with staurosporine for 30 min. To measure transient Ca 2ϩ i responses elicited by neomycin, the buffer solution was devoid of MgCl 2 , CaCl 2 , 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 Ca 2ϩ i response at a given concentration of the agonist was determined using the following method. If the peak increases in Ca 2ϩ 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 Ca 2ϩ i response (Rn) at any given agonist concentration (Cn) is defined as the sum, P1 ϩ P2 ϩ P3ϩ . . . ϩPn. The responsiveness of the wild type and mutant receptors to agonists were compared by determining both EC 50 values and the maximal responses of the respective CaRs. EC 50 has been defined as the effective concentration of an agonist giving half of the maximal Ca 2ϩ i response and was determined by plotting the concentration-response curve. The cumulative maximal Ca 2ϩ i response has been defined as the cumulative Ca 2ϩ i response at the highest agonist concentration achieved by the last addition.
Statistical Analysis-The mean EC 50 for the wild type or each mutant receptor in response to increasing concentrations of Ca 2ϩ o or other CaR agonists was calculated from the EC 50 values for all of the individual experiments and is expressed with the S.E. as the index of dispersion. Comparisons of the EC 50 values were performed using analysis of variance or Duncan's multiple comparison test (24). A p value of Յ0.05 was considered to indicate a statistically significant difference.
Crude Plasma Membrane Preparations from Transfected HEK293 Cells-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. (25). Confluent cultured cells in 100-mm culture plates were rinsed twice with phosphate-buffered saline and treated with 0.02% EDTA in phosphatebuffered saline at 37°C for 5 min. The detached cells were pelleted and suspended in 300 l 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 g/ml aprotinin, 30 g/ml leupeptin, 1 mg/ml Pefabloc, 50 g/ml calpain inhibitor, 50 g/ml bestatin, and 5 g/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 ϫ g for 20 min to remove nuclei and mitochondria. The supernatant was subsequently sedimented at 43,500 ϫ 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.
Western Analysis of Plasma Membrane Proteins-After determination of protein concentrations in the crude plasma membrane preparations using the Pierce BCA protein assay, an appropriate amount of membrane protein (4 g) was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (26). 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

Ca 2ϩ
i Dynamics in Cells Transfected with the CaR primary anti-CaR antibody (4641) (20) and then with a secondary, goat anti-rabbit antibody conjugated to horseradish peroxidase (Sigma, diluted 1:500). The Ca 2ϩ o -sensing receptor protein was detected with an ECL system (Amersham Pharmacia Biotech).
Immunoprecipitation of Flag-tagged CaRs-HEK293 cells transiently transfected with receptors were rinsed twice with phosphatebuffered 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 g/ml aprotinin, 30 g/ml leupeptin, 1 mg/ml Pefabloc, 50 g/ml calpain inhibitor, 50 g/ml bestatin, and 5 g/ml pepstatin (1ϫ 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 g of monoclonal anti-Flag M2 antibody (VWR Scientific), 400 l of H 2 O, 500 l of 2ϫ immunoprecipitation buffer, and 100 l of total lysate containing 500 g of protein were added. The mixture was incubated at 4°C for 1 h. To the mixture was added 5 l 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 l of 10% protein A-agarose (Life Technologies, Inc.) for an additional 3-h incubation at 4°C. The immunoprecipitates were washed three times with 1ϫ immunoprecipitation buffer and twice with phosphate-buffered saline containing protease inhibitors as described above. After one additional wash with 50 l of PKC assay dilution buffer (20 mM MOPS, pH 7.2, 25 mM ␤-glycerol phosphate, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 1 mM CaCl 2 ) purchased from Upstate Biotechnology, the samples were ready for in vitro phosphorylation.
In  i responses is shown in Fig. 1A. In cells treated with 100 nM PMA, the Ca 2ϩ i responses at low Ca 2ϩ o concentrations (1.5-4.5 mM) were markedly attenuated ( Fig. 1B) i responses in cells transfected with the CaR (Fig. 1C) were solely the result of release of Ca 2ϩ from intracellular stores, and these responses were not present in cells mock-transfected with vector alone (data not shown). Neomycin-elicited Ca 2ϩ i responses were substantially attenuated by pretreatment with PMA (1 M) (Fig. 1D) at all concentrations of neomycin tested, and the maximal cumulative Ca 2ϩ i response was reduced to 25% of the control. PMA treatment increased the EC 50 Fig. 1, A and B. Thus the cumulative transient Ca 2ϩ i responses were markedly affected by PMA (Fig. 4A). In contrast, the sustained Ca 2ϩ i responses were similar in control and PMA-treated cells (Fig. 4B). In cells mock-transfected with vector alone, there was also a gradual Ca 2ϩ i increase. However, at least 60% of this increase resulted

Ca 2ϩ
i Dynamics in Cells Transfected with the CaR from leakage of fura-2 over the ϳ20-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 Ca 2ϩ i responses in the receptor-transfected cells were significantly higher than those in vectortransfected cells.
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 CaRevoked Ca 2ϩ i responses (Fig. 5) i is through activation of PKC. We next determined which residues are involved in PKCmediated regulation of the CaR by performing site-directed mutagenesis on the five predicted PKC sites. One of these sites, Thr 646 , is located in the first intracellular loop; another one, Ser 794 , is in the third intracellular loop; and the remainder (Thr 888 , Ser 895 , and Ser 915 ) are in the cytoplasmic tail. The expression of these mutant receptors were then examined by Western analysis, as described previously (20). After transient transfection, crude plasma membrane proteins were isolated and subjected to reduced 4 -12% SDS-PAGE, and the CaRs were detected using a specific anti-CaR antibody. In Fig. 6, the two bands between 140 and 200 kDa are monomeric forms of the CaR; the bands above 200 kDa are also specific for the CaR and are not present in the vector-transfected cells, as shown previously (20) (see also Fig. 9). None of the mutations substantially altered the expression level of the receptor.
The activity of each mutant receptor was examined by measuring its Ca 2ϩ i responses to CaR agonists. The mutant recep- o ] of the CaR, as shown in Fig. 7 and Table I o ] similar to that of the triply mutated receptor.
In order to determine which PKC sites are responsible for the inhibitory effects of PKC activators on CaR agonist-evoked increases in Ca 2ϩ i , cells transfected with mutant receptors were pretreated with one of the PKC activators, 100 nM PMA, for 1-3 min. The patterns of the Ca 2ϩ i responses for receptors with the single point mutations, T646V, S794A, S895A, or S915A (data not shown), or with the double mutation, S895A/ S915A (data not shown), were similar to that of the wild type receptor (Fig. 1B). PMA markedly attenuated low Ca 2ϩ o -elicited Ca 2ϩ i responses mediated by these mutant receptors. As shown in Fig. 7 and Table I o ] values of all of the mutant receptors containing T888V. The receptor with T888V alone showed some decrease in its Ca 2ϩ i responses at 1.5, 2.5, and 3.5 mM Ca 2ϩ o (data not shown). Receptors with one more mutation in addition to Thr 888 , such as T888V/S895A and T888V/S915A, did not show any alterations in the pattern of the Ca 2ϩ i responses observed with the mutant receptor with T888V alone (data not shown). However, receptors with two or more additional mutations, such as the triply mutated (T888V/S895A/S915A) and quintuply mutated CaRs, exhibited markedly reduced inhibitory effects of PMA, only showing some decrease in the Ca 2ϩ i response at 1.5 mM Ca 2ϩ o (data not shown). Thus, the effect of PKC activation by PMA on CaR-mediated increases in Ca 2ϩ i was mostly mediated by one PKC site, Thr 888 .
To demonstrate that the effect of staurosporine on the wild type CaR was, at least in part, the result of prevention of PKC-induced phosphorylation of the receptor, we examined the effect of staurosporine on the mutant receptors carrying PKC site mutations. As shown in Fig. 7 and Table I o ] values upon treatment with PMA, were least affected by treatment with staurosporine. Fig. 7 and Table I also show that the pretreatment of staurosporine prevented the inhibitory effects of PMA on the Ca 2ϩ i responses of triply and quintuply mutated receptors, similar to what we observed with the wild type receptor. In summary, the presence of the T888V mutation abolished most of modulatory effects of PKC on the function of the CaR.
To define further the effect of mutations at PKC phosphorylation sites on CaR-evoked release of intracellular Ca 2ϩ stores, the CaR-induced influx of Ca 2ϩ o was prevented by using neomycin as an agonist in the presence of 1 mM EGTA and in the absence of Ca 2ϩ o and Mg 2ϩ o , as before. Neomycin elicited substantial Ca 2ϩ i responses in cells that had been transfected with all of the mutant receptors in the absence of PMA. In cells that a Significantly (p Յ 0.05) different from controls. b Significantly (p Յ 0.05) different from the wild type control.
FIG. 6. Western analysis to assess expression of the wild type and mutant receptors. Crude plasma membrane proteins (4 g) isolated from CaR-transfected HEK293 cells were subjected to a 4 -12% gradient SDS-PAGE in the order (from left to right): wild type CaR, T646V, S794A, T888V, S895A, S915A, T888V/S895A, T888V/S915A, S895A/S915A, triply mutated CaR (T888V/S895A/S915A), and quintuply mutated CaR. The receptor proteins were detected with a specific anti-CaR antiserum, 4641, as described under "Experimental Procedures." The blot shown is a representative of the pattern seen in two protein preparations from two independent transfections. i responses are shown in Fig. 8. Although the maximal cumulative responses of the T888V and quintuply mutated receptors in the presence of PMA were reduced to about 50 and 60% of the control, respectively, both receptors (Fig. 8, B and D) exhibited substantially greater responses than that of the wild type receptor (Fig. 1D) in the presence of PMA. Thus, the mutation T888V blocked most of the effect of PKC on CaR-induced mobilization of intracellular Ca 2ϩ stores In vitro phosphorylation showed that the bands at 140 and 160 kDa and higher molecular masses immunopurified from cells transfected with the Flag-tagged wild type receptor were substantially labeled with 32 P (Fig. 9A, lane 1). These bands were absent in the sample isolated from the vector-transfected cells, indicating that they were CaR-specific (Fig. 9A, lane 5). The mutant receptor with the two mutations, T646V/S794A, and the quadruply mutated receptor, T646V/S794A/S895A/ S915A, were phosphorylated similarly to the wild type receptor (Fig. 9A, lanes 2 and 3). Moreover, the CaR-specific bands for these two mutant receptors and the wild type CaR were all phosphorylated at levels 3-4 times higher than the mutant receptor carrying mutations in all five PKC sites based on densitometric analysis (Fig. 9A, lane 4). In contrast, the nonspecific bands at their respective positions (i.e. those present in both CaR-and vector-transfected cells) had similar phosphorylation intensities in all samples (Fig. 9A, lanes 1-5). In addition, Fig. 9B shows that the immunoreactivities of the CaRspecific bands in Fig. 9A, lanes 1-4, were similar for all of the receptors when we detected with anti-CaR antibody, 4641, and are absent in Fig. 9B, lane 5, i.e. the vector control. Therefore, in conclusion, we have established that Thr 888 is the major site of the CaR that is phosphorylated by PKC in vitro. Moreover, this site mediates the PKC-induced uncoupling of the receptor from release of intracellular Ca 2ϩ stores. i responses in these cells. In the present study, we found that these effects can be largely eliminated by mutating Thr 888 in the CaR. These results also suggest that this site may have been partially phosphorylated by PKC under our standard experimental conditions; therefore, the activity of the CaR can potentially be modulated by either activating or inhibiting PKC in vivo, in agreement with previous studies in bovine parathyroid cells (6).
Activation of PKC by PMA mainly blocked the CaR-mediated Ca 2ϩ i responses resulting from mobilization of Ca 2ϩ from intracellular stores in cells transfected with the wild type receptor or with mutant CaRs in which the PKC site, Thr 888 , was preserved. This PMA-induced inhibition of CaR-mediated Ca 2ϩ release is particularly apparent with the use of an alternative CaR agonist, neomycin, in the absence of extracellular Ca 2ϩ , when uptake of Ca 2ϩ o was totally eliminated (Fig. 1D). We were able to block substantially the inhibitory effect of PKC activation on CaR-induced release of Ca 2ϩ stores by mutating threonine at position 888. Therefore, PKC-mediated phosphorylation of the CaR at threonine 888 markedly uncouples the receptor from release of Ca 2ϩ from its intracellular stores.
Phosphorylation at position 888 in all of the CaRs preserving Thr 888 had little impact on Ca 2ϩ o -elicited Ca 2ϩ i responses at 5.5 mM or higher concentrations of Ca 2ϩ o (e.g. Fig. 1, A and B Table I). The S.E. is indicated with a vertical line at the top of each bar.

Ca 2ϩ i Dynamics in Cells Transfected with the CaR
Ca 2ϩ influx (Fig. 1B). Consistent with the hypothesis that PMA has little or no impact on high Ca 2ϩ o -stimulated, CaR-mediated Ca 2ϩ influx, PMA had no effect on CaR activation-dependent sustained Ca 2ϩ i increases, which are presumably maintained by CaR-induced increases in Ca 2ϩ influx and/or decreases in Ca 2ϩ efflux. Nevertheless, it is premature to con-clude that PKC phosphorylation at position 888 has no impact on CaR-stimulated Ca 2ϩ influx, because it is hard to distinguish between the contributions of extracellular and intracellular sources of calcium to increases in Ca 2ϩ i , when extracellular calcium is employed as an agonist for the CaR. To elucidate fully the effects of PMA, if any, on CaR-activation dependent Ca 2ϩ influx, further studies are needed to measure directly Ca 2ϩ influx.
CaR activation-dependent Ca 2ϩ influx may be mediated, in part, by Ca 2ϩ -permeable, nonselective cation channels that we have observed in both parathyroid cells (28) and HEK293 cells (29). CaR agonists (neomycin and Ca 2ϩ o ) significantly increase the probability of channel opening in HEK293 cells stably transfected with the CaR but not in nontransfected HEK293 cells that do not express the CaR (29). Thus, the enhanced activity of Ca 2ϩ -permeable nonselective cation channels in CaR-transfected HEK293 cells could contribute to the sustained increases in Ca 2ϩ i in the presence of CaR agonists. These nonselective cation channels are also thought to contribute to influx of Ca 2ϩ o into hippocampal neurons and to regulation of their excitation (30,31).
In PMA-treated cells transfected with the wild type CaR, there was still some residual Ca 2ϩ i response (ϳ25%) to neomycin, even in the total absence of Ca 2ϩ influx. A substantially higher concentration of neomycin (350 versus 150 M in the absence of PMA) was required to elicit the initial response, consistent with observations made earlier in this laboratory (6). That is, activators of PKC inhibited by 50 -60% the high Ca 2ϩ o -stimulated generation of inositol phosphates in CaRexpressing bovine parathyroid cells and reduced inositol trisphosphate levels at low Ca 2ϩ o , presumably by reducing turnover of phosphoinositides by phospholipase C. It is possible that PMA selectively uncouples the receptor from one subtype of G-protein but not another, both of which activate phospholipase C. In rat portal vein myocytes, G q and G 11 have been

Ca 2ϩ
i Dynamics in Cells Transfected with the CaR shown to have distinct functions in coupling ␣ 1 -adrenoreceptors to Ca 2ϩ release and Ca 2ϩ entry (32). In this system, it appeared that G q activated hydrolysis of phosphatidylinositol 4,5-bisphosphate with an attendant release of Ca 2ϩ from inositol trisphosphate-sensitive intracellular stores, whereas G 11 enhanced Ca 2ϩ influx. By analogy, it is possible that phosphorylation of threonine at amino acid position 888 uncouples the CaR from G q but not G 11 , thereby largely inhibiting release of Ca 2ϩ from intracellular stores but not Ca 2ϩ influx. When we mutated all five predicted PKC sites in the CaR, there were still some residual effects of the PKC activator (PMA) and inhibitor (staurosporine) on the quintuply mutated receptor. For example, PMA reduced the maximal cumulative Ca 2ϩ i response to neomycin by 40%. Nevertheless, activation of PKC did not change the EC 50 of the Ca 2ϩ i response to neomycin. It is possible, therefore, that PKC activation may phosphorylate some additional PKC site(s) on the receptor or PKC sites on other components in the signal transduction pathway. Alternatively, PMA may reduce store capacity as has been shown in NIH 3T3 cells (33).
In summary, we have demonstrated that PKC modulation of CaR-mediated Ca 2ϩ i responses is primarily mediated by Thr 888 , which can be phosphorylated by PKC in vitro. Phosphorylation at Thr888 inhibits most of the agonist-induced increases in Ca 2ϩ i due to release from intracellular stores. Because PKC activation blocks the high Ca 2ϩ o -induced suppression of PTH secretion (5, 7, 8, 10 -13, 34, 35) in parathyroid cells, we postulate that the phospholipase C/inositol trisphosphate pathway, leading to release of Ca 2ϩ from stores and/or other downstream effects, is associated with PKC regulation of PTH secretion. In addition, regulation of PKC activity in vivo provides a means of modulating the function of the CaR and, ultimately, calcium homeostasis.