Residue 826 in the Calcium-Sensing Receptor Is Implicated in the Response to Calcium and to R-568 Calcimimetic Compound (original) (raw)
Related papers
Calcimimetic R-568 effects on activity of R990G polymorphism of calcium-sensing receptor
Journal of Molecular Endocrinology, 2010
Previous studies have demonstrated a gain-of-function of the calcium-sensing receptor (CASR) gene R990G polymorphism. In this study, activation of the R990G CASR stably transfected in HEK-293 (HEK-990G) cells compared with that of the common variant (HEK-wild-type (WT)) by increasing concentrations of CaCl 2 or calcimimetic R-568 caused significantly higher intracellular free calcium concentration ([Ca 2C ] i ) and lower Ca-EC 50 . Moreover, the [Ca 2C ] i oscillation percentage was higher with a larger sinusoidal pattern in HEK-990G. R-568 induced a shift of the oscillatory events from 4 to 2 mmol/l extracellular calcium concentration in HEK-990G cells and increased the sinusoidal oscillation percentage in comparison with HEK-WT. Preincubation with thapsigargin or phospholipase C inhibitors completely prevented oscillations in both cell lines, consistent with the involvement of the inositol trisphosphate pathway, while protein kinase C inhibitor prevented oscillations in HEK-WT cells only. Finally, CaCl 2 and R-568 caused a significant increase in p44/42 extracellular signaling-regulated kinase phosphorylation, with the mean Ca-EC 50 values being significantly lower in HEK-990G. Our findings demonstrated that the 990G allele is associated with high sensitivity to R-568, which provided new evidence for differences in CASR signaling.
2007
Cinacalcet, a novel calcimimetic compound, is effective in reducing parathyroid hormone (PTH) levels in approximately 70% of patients with secondary hyperparathyroidism. However, interindividual variations in the dose required to achieve the treatment goal have been noted in clinical studies. Our investigation examined the genetic polymorphisms of the calcium-sensing receptor (CaSR) gene as one possible cause of the different responses to cinacalcet. We report data on seven end-stage renal failure patients who were treated with regular haemodialysis and who participated in clinical trials of cinacalcet. All patients had secondary hyperparathyroidism with baseline intact PTH (iPTH) levels greater than 600 pg/ml. Three patients were male and four female with a mean ± SD age of 60 ± 12 years. DNA was extracted from peripheral lymphocytes. An area in exon 7 of the CaSR gene was amplified by the polymerase chain reaction and sequenced. Mean ± SD baseline iPTH was 1086 ± 189 pg/ml. The five patients without Arg990Gly demonstrated a 29.7 ± 4.0% (± SEM) reduction in iPTH from individual baseline. One patient was found to be homozygous for the Arg990Gly polymorphism and another was heterozygous for both arginine and glycine alleles. The homozygous patient showed a significantly higher sensitivity to cinacalcet compared to the other patients (P = 0.003) with a 76.3 ± 7.7% reduction in iPTH from baseline. No polymorphisms were noted in codons 986 or 1011. This preliminary study points to the possibility that patients homozygous for glycine at the 990 position in exon 7 of the CaSR may be more sensitive to the calcimimetic drug cinacalcet compared to those who are homozygous for arginine at that location. Pharmacogenetics and Genomics 15:29-34 c 2005 Lippincott Williams & Wilkins.
The Journal of Clinical Endocrinology and Metabolism, 2008
Context and Objective: Inactivating mutations in the calcium-sensing receptor (CaSR) gene cause neonatal severe hyperparathyroidism and familial hypocalciuric hypercalcemia (FHH). The aims of the present study were the functional characterization of novel mutations of the CaSR found in FHH patients, the comparison of in vitro receptor function with clinical parameters, and the effect of the allosteric calcimimetic NPS R-568 on the signaling of mutant receptors. Methods: Wild-type and mutant CaSRs (W530G, C568Y, W718X, M734R, L849P, Q926R, and D1005N) were expressed in human embryonic kidney 293 cells. Receptor signaling was studied by measuring intracellular free calcium in response to different concentrations of extracellular calcium (͓Ca 2ϩ ͔ o). Results: Four CaSR mutations (C568Y, W718X, M734R, and L849P) demonstrated a complete lack of a ͓Ca 2ϩ ͔ o-induced cytosolic Ca 2ϩ response up to 30 mM ͓Ca 2ϩ ͔ o , whereas the CaSR mutants W530G, Q926R, and D1005N retained some sensitivity to ͓Ca 2ϩ ͔ o. There was no significant relation between the in vitro calcium sensitivity, serum calcium, and intact PTH levels in the patients. Patients with C-terminal CaSR mutations had a calcium to creatine ratio above the established diagnostic threshold of 0.01 for FHH. The calcimimetic NPS R-568 enhanced the responsiveness to ͓Ca 2ϩ ͔ o in CaSR mutants of the extracellular domain (W530G and C568Y) as well as the intracellular C-terminal domain (Q926R and D1005N). Conclusion: Therefore, calcimimetics might offer medical treatment for symptomatic FHH patients, and more important, for patients with neonatal severe hyperparathyroidism that harbor calcimimetic-sensitive CaSR mutants.
European Journal of Endocrinology, 2013
ObjectiveGain-of-function mutations of the calcium-sensing receptor (CASR) gene have been identified in patients with sporadic or familial autosomal dominant hypocalcemia (ADH). Inactivating mutations of the CASR gene cause familial hypocalciuric hypercalcemia (FHH). Here, we report two novel CASR mutations affecting the same amino acid (p.N802); one causes ADH and the other atypical FHH.Patients and methodsThe first patient, an 11-year-old girl suffering from hypocalcemia, developed nephrocalcinosis when she was only 5 years old. The second patient is a 30-year-old woman who presented with mild hypercalcemia. PCR amplification of CASR coding exons and direct sequencing of PCR products were used to identify mutations. Site-directed mutagenesis was used to generate mutated CASR cDNAs in an expression plasmid. Using the MAPK assay system and transient transfection of Cos-7 cells with wild-type (WT) and mutated CASR, we studied the responses of these mutated receptors to extracellular ...
The Journal of Clinical Endocrinology & Metabolism, 2013
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...
Pharmacology of the calcium sensing receptor
Clinical cases in mineral and bone metabolism : the official journal of the Italian Society of Osteoporosis, Mineral Metabolism, and Skeletal Diseases, 2013
Calcium sensing receptor (CASR) is a G-protein couple receptor which plays a key role in calcium homeostasis in vertebrates. Its extracellular domain is sensitive to divalent cations, aminoacids and polyamines. In parathyroid glands, CASR activation causes parathyroid hormone (PTH) reduction and subsequently a decrease in blood calcium concentration. In PTH-dependent disorders, e.g. primary and secondary hyperparathyroidism (HPT), the need for therapeutic options other than surgery led to the synthesis of various allosteric CASR agonists (calcimimetics), such as cinacalcet. Cinacalcet is the only calcimimetic approved for HPT secondary to chronic kidney disease (CDK), parathyroid carcinoma, and, in some countries, primary HPT. Clinical trials showed that cinacalcet reduced PTH and calcemia both in CDK and primary HPT, lowering the risk of bone fractures, surgery, and cardiovascular complications in the former patients. Long-term safety and pharmacoeconomics have to be fully tested y...
Evidence in Favor of a Calcium-Sensing Receptor in
2015
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Journal of Biological Chemistry, 2002
e ) induces intracellular calcium (Ca 2؉ i ) oscillations that are dependent on thapsigargin-sensitive intracellular calcium stores. Phenylalkylamines such as NPS R-568 are allosteric modulators (calcimimetics) that activate CaR by increasing the apparent affinity of the receptor for calcium. We determined, by fluorescence imaging with fura-2, whether the calcimimetic NPS R-568 could activate Ca 2؉ i oscillations in HEK-293 cells expressing human CaR. NPS R-568 was more potent than Ca 2؉ e at eliciting Ca 2؉ i oscillations, particularly at low [Ca 2؉ ] e (as low as 0.1 mM). The oscillation frequencies elicited by NPS R-568 varied over a 2-fold range from peak to peak intervals of 60 -70 to 30 -45 s, depending upon the concentrations of both Ca 2؉ e and NPS R-568. Finally, NPS R-568 induced sustained (>15 min after drug removal) Ca 2؉ i oscillations, suggesting slow release of the drug from its binding site. We exploited the potency of NPS R-568 for eliciting Ca 2؉ i oscillations for structural studies. Truncation of the CaR carboxyl terminus from 1077 to 886 amino acids had no effect on the ability of Ca 2؉ or NPS R-568 to induce Ca 2؉ i oscillations, but further truncation (to 868 amino acids) eliminated both highly cooperative Ca 2؉ -dependent activation and regular Ca 2؉ i oscillations. Alanine scanning within the amino acid sequence from Arg 873 to His 879 reveals a linkage between the cooperativity for Ca 2؉ -dependent activation and establishment and maintenance of intracellular Ca 2؉ oscillations. The amino acid residues critical to both functions of CaR may contribute to interactions with either G proteins or between CaR monomers within the functional dimer.