ObjectiveThe calcium-sensing receptor (CASR) is a key controller of calcium homeostasis by regulating parathyroid hormone (PTH) secretion and renal calcium reabsorption. CASRT888 is a protein kinase C (PKC) phosphorylation site in the receptor's intracellular domain that has previously been identified as a critical negative regulator of CASR downstream signaling in vitro, but whose importance in vivo is unknown.Case reportThe proband presented with mild symptomatic hypocalcemia following treatment for nephrotic syndrome due to minimal change glomerulonephropathy. Laboratory tests revealed inappropriately normal PTH concentrations and relative hypercalciuria typical of autosomal dominant hypocalcemia. His asymptomatic father had similar laboratory test results.Design and methodsThe CASR gene was sequenced. To investigate the molecular consequences of CASRT888M mutation, site-directed mutagenesis was used to modify the wild-type (wt)-CASR gene, with the resulting mutant being transfected transiently into HEK-293 cells.ResultsA novel CASR missense mutation, T888M, was identified in both cases. The CASRT888M mutant exhibited enhanced sensitivity to extracellular calcium concentration, both for intracellular calcium (Ca2+i) mobilization and for ERK phosphorylation, despite having unaltered levels of cell surface expression. Furthermore, CASRT888M elicited sustained Ca2+i mobilization rather than high frequency Ca2+i oscillations, and, unlike the wt-CASR, the response was resistant to acute inhibition by the PKC activator, phorbol 12-myristate 13-acetate.ConclusionsThe clinical and functional data provide the first genotype–phenotype correlation for a mutation at T888, indicating its critical physiological importance in CASR signaling. Thus, CASRT888 represents a functionally important, inhibitory phosphorylation site that contributes to the control of PTH secretion.
Protein Kinase C Modulates Agonist-sensitive Release of Ca2+from Internal Stores in HEK293 Cells Overexpressing the Calcium Sensing Receptor
