Dominant negative effect of the extracellular domain of CASR

The role of the calcium-sensing receptor (CASR) in the skeleton is unclear. CASR null mice (CASR -/- ), created by deletion of exon 5 and disruption of the extracellular domain (ED), have no bone phenotype after rescue of hyperparathyroidism. In contrast, the conditional deletion in osteoblasts of exon 7 (CASR ∆exon7 ), which encodes the 7-transmembrane and C-terminal domains, results in severe skeletal abnormalities. The disparities between these models could be explained by a hypomorphic mutation in CASR -/- mice or by the secretion of a dominant negative ED by CASR ∆exon7 mice. A hypomorphic mutation seems unlikely, since deletion of exon 5 disrupts CASR function in vitro and results in a prenatal lethal phenotype in vivo. We tested the alternative possibility that the ED of CASR acts as dominant negative secreted protein. Transfection of CASR 1-464 (encoding exons 2-5) cDNA into HEK293 cells resulted in the secretion of the predicted N-terminal CASR protein. Recombinant CASR 1-464 resulted in a dose-dependent and specific inhibition of extracellular calcium-mediated CASR signaling in vitro and increased serum PTH levels in vivo. These results suggest that the extracellular domain of CASR has biological activity in vitro and in vivo and may contribute to off target effects in conditional CASR ∆exon7 mice.