Increased receptor stimulation elicits differential calcium-sensing receptor(T888) dephosphorylation.
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).
Faculty of Life Sciences, The University of Manchester, Manchester M13 9NT, United Kingdom.
Wanda D
WD
McCormick
Rebecca
R
Atkinson-Dell
Katherine L
KL
Campion
Hee-Chang
HC
Mun
Arthur D
AD
Conigrave
Donald T
DT
Ward
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002417:Cattle; D002478:Cells, Cultured; D006801:Humans; D015151:Immunoblotting; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D010280:Parathyroid Glands / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D054648:Protein Phosphatase 2 / Q000378:metabolism; D044169:Receptors, Calcium-Sensing / Q000378:metabolism*; D015398:Signal Transduction
D010281:Parathyroid Hormone; D044169:Receptors, Calcium-Sensing; D011493:Protein Kinase C; D054648:Protein Phosphatase 2; D002118:Calcium