scholarly journals Identification and functional characterization of loss-of-function mutations of the calcium-sensing receptor in four Italian kindreds with familial hypocalciuric hypercalcemia

2009 ◽  
Vol 160 (3) ◽  
pp. 481-489 ◽  
Author(s):  
Filomena Cetani ◽  
Monica Lemmi ◽  
Davide Cervia ◽  
Simona Borsari ◽  
Luisella Cianferotti ◽  
...  
Keyword(s):  
Cell Surface ◽  
Functional Characterization ◽  
Extracellular Calcium ◽  
Dominant Negative Effect ◽  
Familial Hypocalciuric Hypercalcemia ◽  
Functional Responses ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing ◽  
Mutant Receptors

ObjectiveIdentification and characterization of calcium-sensing receptor (CASR) mutations in four unrelated Italian kindreds with familial hypocalciuric hypercalcemia.DesignClinical evaluation and genetic analysis ofCASRgene. Functional characterization of mutatedCASRs.MethodsDirect sequencing ofCASRgene in genomic DNA. Studies ofCASR-mediated increases in cytosolic calcium concentration [Ca2+]iinCASR-transfected COS-7 cellsin vitro.ResultsFour unreported heterozygousCASRmutations were identified, including three missense (H595Y, P748H, and C765W) and one splice site (IVS2+1G>C) mutation. The H595Y, P748H, and C765W mutant receptors, although expressed at normal levels on the cell surface, showed a reduced response in [Ca2+]irelative to the wildtype (WT)CASRto increasing extracellular calcium concentrations. Cotransfection experiments showed that the H595Y and P748H mutants did not affect the apparent affinity of the WTCASRfor calcium, suggesting that they do not exert a dominant-negative effect. On the other hand, the co-transfected C765W mutant decreased the maximum response of the WTCASRto calcium, suggesting that it may reduce the effective concentration of the normalCASRon the cell surface or impair its maximal signaling capacity.ConclusionsFourCASRmutations were identified. The reduced functional responses to extracellular calcium and normal expression of the mutant receptors suggest that conformational changes account for alteredCASRactivity. Moreover, a reduced complement of normalCASRsin these heterozygous patients, perhaps combined with a mutant receptor-induced decrease in maximal activity of the WT receptor, may contribute to defective calcium-sensingin vivo.

scholarly journals Identification and Functional Characterization of a Calcium-Sensing Receptor Mutation in an Infant with Familial Hypocalciuric Hypercalcemia

2016 ◽  
Vol 8 (3) ◽  
pp. 341-346 ◽  
Author(s):  
Anna Papadopoulou ◽  
Evangelia Gole ◽  
Katerina Melachroinou ◽  
Christos Meristoudis ◽  
Tania Siahanidou ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Familial Hypocalciuric Hypercalcemia ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing

scholarly journals 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

2013 ◽  
Vol 98 (10) ◽  
pp. E1692-E1701 ◽  
Author(s):  
Akie Nakamura ◽  
Tomoyuki Hotsubo ◽  
Keiji Kobayashi ◽  
Hiroshi Mochizuki ◽  
Katsura Ishizu ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Allosteric Modulators ◽  
Familial Hypocalciuric Hypercalcemia ◽  
Calcium Sensing Receptor ◽  
Activating Mutations ◽  
Casr Gene ◽  
Calcium Sensing ◽  
Inactivating Mutations

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.

scholarly journals Novel Activating Mutations of the Calcium-Sensing Receptor: The Calcilytic NPS-2143 Mitigates Excessive Signal Transduction of Mutant Receptors

2010 ◽  
Vol 24 (9) ◽  
pp. 1888-1888
Author(s):  
Saskia Letz ◽  
Ramona Rus ◽  
Christine Haag ◽  
Helmuth-Günther Dörr ◽  
Dirk Schnabel ◽  
...  
Keyword(s):  
Autosomal Dominant ◽  
Functional Characterization ◽  
Calcium Excretion ◽  
Wild Type ◽  
Calcium Sensing Receptor ◽  
Activating Mutations ◽  
Calcium Sensing ◽  
Mutant Receptors ◽  
Autosomal Dominant Hypocalcemia

Abstract Context and Objective: Activating mutations in the calcium-sensing receptor (CaSR) gene cause autosomal dominant hypocalcemia (ADH). The aims of the present study were the functional characterization of novel mutations of the CaSR found in patients, the comparison of in vitro receptor function with clinical parameters, and the effect of the allosteric calcilytic NPS-2143 on the signaling of mutant receptors as a potential new treatment for ADH patients. Methods: Wild-type and mutant CaSR (T151R, P221L, E767Q, G830S, and A844T) were expressed in human embryonic kidney cells (HEK 293T). Receptor signaling was studied by measuring intracellular free calcium in response to different concentrations of extracellular calcium ([Ca2+]o) in the presence or absence of NPS-2143. Results: All ADH patients had lowered serum calcium ranging from 1.7 to 2.0 mm and inadequate immunoreactive PTH and urinary calcium excretion. In vitro testing of CaSR mutations from these patients revealed exaggerated [Ca2+]o-induced cytosolic Ca2+ responses with EC50 values for [Ca2+]o ranging from 1.56 to 3.15 mm, which was lower than for the wild-type receptor (4.27 mm). The calcilytic NPS-2143 diminished the responsiveness to [Ca2+]o in the CaSR mutants T151R, E767Q, G830S, and A844T. The mutant P221L, however, was only responsive when coexpressed with the wild-type CaSR. Conclusion: Calcilytics might offer medical treatment for patients with autosomal dominant hypocalcemia caused by calcilytic-sensitive CaSR mutants.

Sign in / Sign up

Export Citation Format

Share Document