title: 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.
abstract: 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.
authors: Department of Pediatrics, Hokkaido University School of Medicine, Sapporo 060-8635, Japan. akieda@med.hokudai.ac.jp.
authors: Akie
authors: A
authors: Nakamura
authors: Tomoyuki
authors: T
authors: Hotsubo
authors: Keiji
authors: K
authors: Kobayashi
authors: Hiroshi
authors: H
authors: Mochizuki
authors: Katsura
authors: K
authors: Ishizu
authors: Toshihiro
authors: T
authors: Tajima
mesh_terms: D002648:Child; D006801:Humans; D006934:Hypercalcemia / Q000151:congenital* / Q000235:genetics; D007011:Hypoparathyroidism / Q000235:genetics*; D007223:Infant; D007231:Infant, Newborn; D009154:Mutation; D044169:Receptors, Calcium-Sensing / Q000235:genetics*
chemical_list: D044169:Receptors, Calcium-Sensing