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).
Highlights
Acute treatment of calcium-sensing receptor (CaR)-HEK cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA) (1 M) increased the immunoreactivity of two protein bands of approximate molecular mass 140 and 160 kDa obtained by immunoblotting using the custom-generated phospho-specific polyclonal antibody raised against CaR residue Thr-888 (Fig. 1), as shown previously (15)
For various times up to 20 min resulting in a significant, timedependent change in the levels of CaRT888 phosphorylation (Fig. 1)
protein kinase C (PKC)-mediated modulation of class C G protein-coupled receptor signaling is emerging as a significant determinant of receptor function for these receptors
Summary
Treatment of CaR-HEK cells with 2 mM Ca2ϩo caused a rapid increase in 160 kDa CaRT888 phosphorylation, reaching significance after only 30 s of exposure (p Ͻ 0.01; Fig. 3A). While cotreatment with 10 mM L-Phe (in the presence of 1.2 mM Ca2ϩo) failed to induce significant CaRT888 phosphorylation (160-kDa mature protein) after 2 min, phosphorylation levels were raised significantly after 10 min, consistent with the more sustained response evoked by 2 mM Ca2ϩo alone (see Fig. 3).
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