A novel mutation of the primary protein kinase C phosphorylation site in the calcium-sensing receptor causes autosomal dominant hypocalcemia

Abstract

Objective: The calcium-sensing receptor (CASR) is a key controller of calcium homeostasis by regulating parathyroid hormone (PTH) secretion and renal calcium reabsorption. CASR T888 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 report: The 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 methods: The CASR gene was sequenced. To investigate the molecular consequences of CASR T888M 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. Results: A novel CASR missense mutation, T888M, was identified in both cases. The CASR T888M mutant exhibited enhanced sensitivity to extracellular calcium concentration, both for intracellular calcium ðCa 2C i Þ mobilization and for ERK phosphorylation, despite having unaltered levels of cell surface expression. Furthermore, CASR T888M elicited sustained Ca 2C i mobilization rather than high frequency Ca 2C i oscillations, and, unlike the wt-CASR, the response was resistant to acute inhibition by the PKC activator, phorbol 12-myristate 13-acetate. Conclusions: The clinical and functional data provide the first genotype-phenotype correlation for a mutation at T888, indicating its critical physiological importance in CASR signaling. Thus, CASR T888 represents a functionally important, inhibitory phosphorylation site that contributes to the control of PTH secretion.

Molecular and clinical insights from studies of calcium-sensing receptor mutations

“…6D). The two C-terminal missense mutations affect residues that are critical for facilitating Ca 2+ i responses (Bai et al 1998, Huang et al 2010, Lazarus et al 2011). The first mutation Val883Met, disrupts a region that is known to interact with calmodulin and dorfin (Huang et al 2010), while the second mutation, Thr888Met, disrupts a protein kinase C phosphorylation site that negatively regulates CaSR coupling to Ca 2+ i stores (Bai et al 1998).…”

Two novel mutations of the calcium-sensing receptor gene affecting the same amino acid position lead to opposite phenotypes and reveal the importance of p.N802 on receptor activity

“…CASR has a large extracellular amino terminal domain (ECD) (606 amino acids corresponding to around 56% of total amino acids), seven transmembrane domains (TMD) (23% of protein), and an intracellular carboxy-terminal domain (ICD) (21% of protein). Known loss-of function mutations are scattered throughout the protein, but gain-of-function mutations are nonrandomly distributed (9): 52.1% map in the ECD (10) and many clusters between residues 116 and 131; 42.3% map in the TMDs with clusters in TMD6 (23.8% of TMD mutants) and in TMD7 (30%); and only 5.6% are in the ICD (11). The identification and analysis of these naturally occurring mutations have helped elucidate the structure and function of this receptor.…”

Novel calcium-sensing receptor cytoplasmic tail deletion mutation causing autosomal dominant hypocalcemia: molecular and clinical study

“…A unique secondary structure, a 15 amino acid a-helical stretch from residue 877-891, may be required for efficient trafficking of the CASR to the cell surface (25). Phosphorylation at T888 by protein kinase C is important for desensitization of the receptor and a mutant (T888M) that is unable to be phosphorylated causes ADH (29).…”