Agonist-Induced Feedback Phosphorylation of the Calcium-Sensing Receptor at the Thr-888 Putative Protein Kinase C Site

Introduction and Aims
Calcium is an essential mineral for numerous physiological processes and extreme changes of blood calcium levels can precipitate life-threatening conditions. Thus, blood Ca²⁺ levels are controlled tightly by the regulated secretion of parathyroid hormone (PTH) and 1,25(OH)₂ vitamin D₃. The calcium-sensing receptor (CaR) is a type III G protein-coupled receptor that senses changes in blood calcium levels ([Ca²⁺]o) so as to allow feedback regulation of PTH secretion. Previous studies have shown that CaR-induced intracellular signalling is affected by activation or inhibition of protein kinase C (PKC) and that CaR residue Thr-888 (CaR_T888), found in the receptor’s cytoplasmic tail, may be the site of PKC regulation. Thus, in the current study we employed a novel phospho-specific antibody to examine directly the phosphorylation of CaR_T888. The first objective was to characterise the specificity of the antibody before studying CaR_T888 phosphorylation in response to phorbol ester (PMA; potent PKC activator) treatment and then CaR activation. Finally, experiments were performed to identify the class of phosphatase most likely responsible for CaR_T888 dephosphorylation.

Methods
CaR_T888 phosphorylation was examined by immunoblotting and immunofluorescence in HEK cells stably transfected with the human parathyroid CaR (CaR-HEK). Cells were grown to 〜80% confluence in DMEM / 10% FBS medium and then exposed to various treatments, at 37^oC for 10 mins, in HEPES buffer containing 0.5mM CaCl₂ prior to lysis in RIPA buffer (immunoblotting) or fixation in paraformaldehyde (immunofluorescence).

Results and Discussion
Incubation of the CaR-HEK cells in PMA elicited increased CaR_T888 phosphorylation and this immunoreactivity was ablated by pre-incubating the antibody with nanomolar concentrations of phospho-peptide but not nonphospho-peptide. This demonstrated the specificity of the antibody and shows that CaR_T888 can be phosphorylated by PMA-sensitive PKC isotypes (i.e. cPKC or nPKC).
　　Next, stimulation of the cells with increased Ca²⁺_o levels or the CaR-selective activator NPS-467R also increased CaR_T888 phosphorylation, indicating the presence of feedback signalling on the CaR. These responses were inhibited by PKC inhibition (GF109203X) or downregulation (chronic PMA pre-treatment).
　　Finally, cells were treated with PMA (to induce CaR_T888 phosphorylation) and then incubated for a further 5 mins in PMA-free buffer (plus GF109203X) during which substantial CaR_T888 dephosphorylation was observed. However, this was prevented by cotreatment with the PP1/PP2A phosphatase inhibitor calyculin A.
　　Together, these data prove that PKC does phosphorylate the CaR upon residue Thr-888, that agonist-induced receptor activation elicits feedback phosphorylation of the receptor, and, that the CaR phosphatase is calyculin-sensitive.