Loss-of-function and gain-of-function mutations of calcium-sensing receptor: functional analysis and the effect of allosteric modulators NPS R-568 and NPS 2143

Objective: Activating mutations in the calcium-sensing receptor (CaSR) gene cause autosomal dominant hypoparathyroidism, and heterozygous inactivating CaSR mutations cause familial hypocalciuric hypercalcemia. Recently, there has been a focus on the use of allosteric modulators to restore the functional activity of mutant CASRs. In this study, the effect of allosteric modulators NPS R-568 and NPS 2143 on CaSR mutants was studied in vitro.

Methods: DNA sequence analysis of the CaSR gene was undertaken in autosomal dominant hypoparathyroidism and familial hypocalciuric hypercalcemia Japanese patients, and the functional consequences for the Gi-MAPK pathway and cell surface expression of CaSR were determined. Furthermore, we studied the effect of NPS R-568 and NPS 2143 on the signal transduction activity and cell surface expression of each mutant CaSR.

Results: We identified 3 activating mutations (S122C, P569H, and I839T) and 2 inactivating mutations (A110T and R172G) in patients. The activating and inactivating mutations caused leftward and rightward shifts, respectively, in the dose-response curves of the signaling pathway. NPS R-568 rescued the signal transduction capacity of 2 inactivating mutants without increasing cell surface expression levels. NPS 2143 suppressed the enhanced activity of the activating mutants without altering cell surface expression levels, although A843E, which is a constitutively active mutant, was suppressed to a lesser degree.

Conclusions: We have identified 4 novel mutations of CaSR. Moreover, our results indicate that allosteric modulators can restore the activity of the loss- and gain-of-function mutant CASRs, identified in this study.