A Ca²⁺-Sensing Receptor Mutation Causes Familial Hypoparathyroidism by Increasing Receptor Sensitivity to Ca²⁺ and Maximal Signal Transduction. • 403

Activating mutations of the Ca²⁺-sensing receptor (CaR) gene cause autosomal dominant hypoparathyroidism (ADHP). The mechanism by which these mutations activate the receptor is not clear. We studied a family with ADHP. The mother and three daughters experienced muscle spasms and/or seizures from early childhood. They were treated with oral calcium and vitamin D analogs. All four patients developed hypercalciuria, nephrocalcinosis and renal insufficiency. A heterozygous missense mutation, F612S, involving the extracellular region of the CaR, was identified in affected family members. The mutation was not present in 50 normal control individuals. We used site-directed mutagenesis to introduce this mutation into the CaR cDNA. Functional expression studies were performed by transfecting HEK-293 cells with wild type (WT) or mutant CaR constructs and measuring the accumulation of radiolabeled inositol phosphates as a function of extracellular Ca²⁺ concentration. Mutant receptor showed a lower EC₅₀ for Ca²⁺ than WT (F612S 2.3 ± 0.06, WT 4.3 ± 0.2 mM, mean ± SEM; p<0.001). In addition, the maximal response to high Ca²⁺ concentrations was significantly increased (F612S 42 ± 3, WT 33± 2 10³-cpm; p<0.02). These changes in receptor function could not be explained by changes in cell surface expression (ELISA, F612S 1.3± 0.6, WT 3.7 ± 0.1 O.D. units). Similarly, Western analysis of expressed receptors showed no evidence for altered protein size or glycosylation. These data suggest that the amino acid substitution affects signal transduction directly. We conclude that CaR mutation F612S causes hypoparathyroidism by increasing both receptor sensitivity to extracellular Ca²⁺ and maximal signal transduction capacity.