Protein kinase C-mediated phosphorylation of the calcium-sensing receptor is stimulated by receptor activation and attenuated by calyculin-sensitive phosphatase activity.
J Biol Chem
; 282(20): 15048-56, 2007 May 18.
Article
in En
| MEDLINE
| ID: mdl-17376781
ABSTRACT
The agonist sensitivity of the calcium-sensing receptor (CaR) can be altered by protein kinase C (PKC), with CaR residue Thr(888) contributing significantly to this effect. To determine whether CaR(T888) is a substrate for PKC and whether receptor activation modulates such phosphorylation, a phospho-specific antibody against this residue was raised (CaR(pT888)). In HEK-293 cells stably expressing CaR (CaR-HEK), but not in cells expressing the mutant receptor CaR(T888A), phorbol ester (PMA) treatment increased CaR(pT888) immunoreactivity as observed by immunoblotting and immunofluorescence. Raising extracellular Ca(2+) concentration from 0.5 to 2.5 mM increased CaR(T888) phosphorylation, an effect that was potentiated stereoselectively by the calcimimetic NPS R-467. These responses were mimicked by 5 mM extracellular Ca(2+) and abolished by the calcilytic NPS-89636 and also by PKC inhibition or chronic PMA pretreatment. Whereas CaR(T888A) did exhibit increased apparent agonist sensitivity, by converting intracellular Ca(2+) (Ca(2+)(i)) oscillations to sustained plateau responses in some cells, we still observed Ca(2+)(i) oscillations in a significant number of cells. This suggests that CaR(T888) contributes significantly to CaR regulation but is not the exclusive determinant of CaR-induced Ca(2+)(i) oscillations. Finally, dephosphorylation of CaR(T888) was blocked by the protein phosphatase 1/2A inhibitor calyculin, a treatment that also inhibited Ca(2+)(i) oscillations. In addition, calyculin/PMA cotreatment increased CaR(T888) phosphorylation in bovine parathyroid cells. Therefore, CaR(T888) is a substrate for receptor-induced, PKC-mediated feedback phosphorylation and can be dephosphorylated by a calyculin-sensitive phosphatase.
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Collection:
01-internacional
Database:
MEDLINE
Main subject:
Parathyroid Glands
/
Phosphoprotein Phosphatases
/
Calcium Signaling
/
Receptors, Calcium-Sensing
/
Enzyme Inhibitors
/
Aniline Compounds
Type of study:
Diagnostic_studies
Limits:
Animals
/
Humans
Language:
En
Journal:
J Biol Chem
Year:
2007
Type:
Article
Affiliation country:
United kingdom