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

• Published in: The journal of clinical endocrinology and metabolism 2013-10, Vol.98 (10), p.E1692-E1701
• Main Authors: Nakamura, Akie, Hotsubo, Tomoyuki, Kobayashi, Keiji, Mochizuki, Hiroshi, Ishizu, Katsura, Tajima, Toshihiro
• Format: Article
• Language: English
• Subjects: Child; Humans; Hypercalcemia - congenital; Hypercalcemia - genetics; Hypoparathyroidism - genetics; Infant; Infant, Newborn; Mutation; Receptors, Calcium-Sensing - genetics
• ISSN: 0021-972X; 1945-7197
• DOI: 10.1210/jc.2013-1974

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.