Increased receptor stimulation elicits differential calcium-sensing receptor(T888) dephosphorylation

Abstract

The calcium-sensing receptor (CaR) elicits oscillatory Ca(2+)(i) mobilization associated with dynamic, inhibitory protein kinase C-mediated phosphorylation of CaR(T888). While modest CaR stimulation elicits Ca(2+)(i) oscillations, greater stimulation either increases oscillation frequency or elicits sustained responses by an unknown mechanism. Here, moderate CaR stimulation (2.5 mm Ca(2+)(o), 10 min) increased CaR(T888) phosphorylation (160-kDa mature receptor) 5-fold in CaR stably transfected HEK-293 cells, whereas 3-5 mm Ca(2+)(o) treatments were without apparent effect. Treatment with 2 mm Ca(2+)(o) caused sustained CaR(T888) phosphorylation (> or = 20 min) and oscillatory Ca(2+)(i) mobilization. However, 5 mm Ca(2+)(o) increased CaR(T888) phosphorylation only briefly while eliciting sustained Ca(2+)(i) mobilization, suggesting that greater CaR activation induces rapid CaR(T888) dephosphorylation, thus permitting sustained Ca(2+)(i) responses. Indeed, 5 mm Ca(2+)(o) stimulated protein phosphatase 2A activity and induced CaR(T888) dephosphorylation following acute phorbol ester pretreatment, the latter effect being mimicked by CaR-positive allosteric modulators (NPS-R467 and l-Phe). Finally, the phosphatase inhibitor calyculin-A reversed CaR-induced inhibition of parathyroid hormone secretion from bovine parathyroid slices and normal human parathyroid cells, demonstrating the physiological importance of phosphorylation status on parathyroid function. Therefore, high Ca(2+)(o)-stimulated protein kinase C acts in concert with high Ca(2+)(o)-induced phosphatase activity to generate and maintain CaR-induced Ca(2+)(i) oscillations via the dynamic phosphorylation and dephosphorylation of CaR(T888).

FIGURE 1.

Phosphorylation of the CaR^T888 residue by PMA in CaR-HEK cells. A, representative immunoblots showing CaR^pT888 (i) and total CaR (ii) immunoreactivity following treatment with 0.5 mm Ca²⁺_o and 1 μm PMA for different lengths of time up to 20 min. B, quantification showing the mean changes in CaR^pT888 expressed in arbitrary (arb.) units (n = 4). **, p < 0.01 versus control by repeated measures ANOVA (Dunnett's post test).

FIGURE 2.

Effect of Ca²⁺_o concentration on CaR^T888 phosphorylation in CaR-HEK cells. A, representative Western blot showing CaR^pT888 (i) and total CaR immunoreactivity (ii) following treatment for 10 min with different concentrations of Ca²⁺_o up to 5 mm. B, quantification of the changes in CaR^T888 phosphorylation expressed in arbitrary (arb.) units for the 160-kDa protein (n = 7). ***, p < 0.001 versus 0.5 mm Ca²⁺_o. ++, p < 0.01 versus 5 mm Ca²⁺_o by repeated measures ANOVA (Tukey post test).

FIGURE 3.

Impacts of a moderate Ca²⁺_o concentration on time-dependent CaR^T888 phosphorylation and Ca²⁺_i mobilization. A, representative Western blot showing CaR^pT888 (i) and total CaR immunoreactivity (ii) following treatment with 2 mm Ca²⁺_o for increasing times up to 20 min. Quantification of the changes in CaR^T888 phosphorylation expressed in arbitrary (arb.) units for the 160-kDa CaR band is shown in panel iii (n = 11). *, p < 0.05, **, p < 0.01 versus t₀ by repeated measures ANOVA (Dunnett's post test). B, representative trace showing Ca²⁺_i changes (Fura-2 ratio) in two single cells (light and dark gray lines) or a “global” cluster of cells (black) in response to 2.5 mm Ca²⁺_o (n = 3).

FIGURE 4.

Impacts of a maximally effective Ca²⁺_o concentration on time-dependent CaR^T888 phosphorylation and Ca²⁺_i mobilization. A, representative Western blot showing CaR^pT888 (i) and total CaR (ii) immunoreactivity following exposure to 5 mm Ca²⁺_o for different lengths of time up to 20 min. Quantification of the changes in CaR^T888 phosphorylation expressed in arbitrary (arb.) units for the 160-kDa CaR band is shown in panel iii (n = 5). **, p < 0.01, ***, p < 0.001 versus t₀; +, p < 0.05, ++, p < 0.01 versus 1-min treatment by repeated measures ANOVA (Tukey post test). B, representative trace showing Ca²⁺_i changes in a single cell (gray) or global cluster of cells (black) in response to 5 mm Ca²⁺_o (n = 3).

FIGURE 5.

Induction of CaR^T888 phosphorylation by the positive allosteric CaR modulators l-Phe and NPS-R467. A, representative immunoblots showing CaR^pT888 (i) and total CaR (ii) immunoreactivity in CaR-HEK cells following treatment with 1.2 mm Ca²⁺_o in the presence or absence of 1 μm NPS-R467 or 10 mm l-Phe for 2, 5, and 10 min. Quantification of the changes in 160 kDa CaR^pT888 immunoreactivity is shown in panel iii (n = 9). *, p < 0.05, **, p < 0.01 (R-467), +p < 0.05 (l-Phe) versus 1.2 mm Ca²⁺_o by one-way ANOVA (Dunnett's post test). Representative traces showing Ca²⁺_i changes in a single cell (gray) or global cluster of cells (black) in response to 1 μm NPS-R467 (Bi) or 10 mm l-Phe (Ci) (in the presence of 1.5 mm Ca²⁺_o). Quantification of the Ca²⁺_i changes is shown as area under the curve/min in panels Bii and Cii (n = 3). *, p < 0.05, ***, p < 0.001 versus responses before and after allosteric treatments, by repeated measures ANOVA. arb., arbitrary; Cont, control.

FIGURE 6.

CaR-induced dephosphorylation of CaR^T888. A, representative Western blots (i) showing anti-CaR^pT888 or total CaR immunoreactivity following a 10-min pretreatment of CaR-HEK cells with 1 μm PMA (including 250 nm GF109203X for the final 30 s) followed by a further 30-s incubation in buffer (250 nm GF109203X) containing either 0.5, 1.2 (± 10 mm l-Phe), 2.2, or 5 mm Ca²⁺_o. Panel ii shows quantification of the relative change in 160 kDa CaR^T888 phosphorylation expressed as a percentage of response in 0.5 mm Ca²⁺_o (n = 10). *, p < 0.05 versus 1.2 mm Ca²⁺_o; +, p < 0.05 versus 1.2 mm Ca²⁺_o and 10 mm l-Phe by ANOVA. B, identical experiment in which PMA-pretreated cells were incubated for the final 30 s in buffer containing 0.5 and 2.2 (± 10 mm l-Phe) or 5 mm Ca²⁺_o (+GF109203X) (n = 8). *, p < 0.05, **, p < 0.01 versus 2.2 mm Ca²⁺_o by repeated measures ANOVA. C, identical experiment in which PMA-pretreated cells were incubated for the final 30 s in buffer containing 2.2 mm Ca²⁺_o (+GF109203X) in the presence or absence of 1 μm NPS-R467. Quantification of the relative change in CaR^T888 phosphorylation expressed as a % of control (cont; n = 11). *, p < 0.05 versus 2.2 mm Ca²⁺_o by paired t test. D, effect of 5 mm Ca²⁺_o on PP2A activity after 2 and 5 min of treatment. *, p < 0.05 by Friedman (n = 7).

FIGURE 7.

Removal of calyculin A permits Ca²⁺_o-induced suppression of PTH secretion. A, bovine parathyroid slices were perifused in 1.2 mm Ca²⁺_o-containing buffer in the presence of 100 nm calyculin A (cal) for 10 min followed by perifusion in the presence or absence of 100 nm calyculin A for 20 min. PTH secretion (quantified by enzyme-linked immunosorbent assay) is shown as % control (control being the mean value for the first 10 min due to the variability of secretion in the absence of its suppression). **, p < 0.01 versus 2–6 min of control by repeated measures ANOVA; +, p < 0.05 versus 26–30 min in continuous calyculin A treatment, by unpaired t test (n = 6). B, bovine parathyroid slices were incubated for up to 30 min in buffer containing 100 nm calyculin A and 1 μm PMA and then homogenized, and their particulate fractions were collected and processed for immunoblotting using CaR^pT888 and total CaR antibodies. *, p < 0.05, **, p < 0.01 versus control (cont) by one-way ANOVA (n = 8). C, in Fura 2-loaded bovine parathyroid cells, the rise in [Ca²⁺]_i elicited by increasing [Ca²⁺]_o from 0.8 to 2 mm, was suppressed by cotreatment with calyculin (100 nm, n = 6). Following washout, exposure to 5 mm Ca²⁺_o demonstrated continued responsiveness of the cells. The three upper traces show representative single cell responses, whereas the lower trace shows the combined response of a cluster of eight cells (global). A.U., arbitrary units. ns, not significant.

FIGURE 8.

Calyculin A attenuates Ca²⁺_o-induced suppression of PTH secretion in normal human parathyroid cells. Human parathyroid cells were prepared and perifused as described under “Experimental Procedures.” Cells were first perifused in 1.2 mm Ca²⁺_o corresponding to the midpoint of the normal range (1.1–1.3 mm). Lowering Ca²⁺_o concentration to 0.8 mm reversibly stimulated PTH secretion, whereas subsequent 1.4 mm Ca²⁺_o exposure suppressed PTH. However, in the continued presence of 1.4 mm Ca²⁺_o, cotreatment with 100 nm calyculin A elicited a progressive increase in PTH secretion to levels similar to or even exceeding those observed at 0.8 mm Ca²⁺_o. Upon the removal of calyculin A, PTH secretion levels slowly returned to their control levels. The result shown is representative of three independent experiments.