Biomedical subjects

Svetlana Pidasheva

Publications and source records attributed to Svetlana Pidasheva.

5 recordsLinked to original sources

PubMed2006

Calcium-sensing receptor dimerizes in the endoplasmic reticulum: biochemical and biophysical characterization of CASR mutants retained intracellularly.

Calcium-sensing receptor (CASR), expressed in parathyroid gland and kidney, is a critical regulator of extracellular calcium homeostasis. This G protein-coupled receptor exists at the plasma membrane as a homodimer, although it is unclear at which point in the biosynthetic pathway dimerization occurs. To address this issue, we have analyzed wild-type and mutant CASRs harboring R66H, R66C or N583X-inactivating mutations identified in familial hypocalciuric hypercalcemia/neonatal severe hyperparathyroid patients, which were transiently expressed in kidney cells. All mutants were deficient in cell signaling responses to extracellular CASR ligands relative to wild-type. All mutants, although as well expressed as wild-type, lacked mature glycosylation, indicating impaired trafficking from the endoplasmic reticulum (ER). Dimerized forms of wild-type, R66H and R66C mutants were present, but not of the N583X mutant. By immunofluorescence confocal microscopy of non-permeabilized cells, although cell surface expression was observed for the wild-type, little or none was seen for the mutants. In permeabilized cells, perinuclear staining was observed for both wild-type and mutants. By colocalization fluorescence confocal microscopy, the mutant CASRs were localized within the ER but not within the Golgi apparatus. By the use of photobleaching fluorescence resonance energy transfer microscopy, it was demonstrated that the wild-type, R66H and R66C mutants were dimerized in the ER, whereas the N583X mutant was not. Hence, constitutive CASR dimerization occurs in the ER and is likely to be necessary, but is not sufficient, for exit of the receptor from the ER and trafficking to the cell surface.

Biophysical Phenomena↗

PubMed2005

Impaired cotranslational processing of the calcium-sensing receptor due to signal peptide missense mutations in familial hypocalciuric hypercalcemia.

The CASR, a cell surface glycoprotein expressed in parathyroid gland and kidney, is critical for maintaining extracellular calcium homeostasis. The inherited disorders, familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT), are caused by inactivating mutations in the CASR gene. The CASR has an N-terminal, 19 amino acid signal peptide that is predicted to direct the nascent polypeptide chain, as it emerges from the ribosome, into the endoplasmic reticulum (ER). Here, we report the functional characterization of three CASR mutations identified in hypercalcemic/hyperparathyroid patients. The mutations, L11S, L13P and T14A, lie within the signal peptide hydrophobic core. When transiently transfected into kidney cells, L11S and L13P mutants demonstrated reduced intracellular and plasma membrane expression and signaling to the mitogen-activated protein kinase pathway in response to extracellular calcium relative to wild-type CASR and the T14A mutant. All mutant CASR RNAs translated into protein normally. In cotranslational processing assays, which test the functionality of the signal peptide in the early secretory pathway, the wild-type CASR and mutant T14A nascent polypeptides were targeted to microsomal vesicles, representing the ER, translocated into the vesicular lumen and underwent core N-glycosylation. In contrast, the L11S and L13P mutants failed to be inserted in the microsomes and undergo glycosylation. This is the first study examining the function of the CASR signal sequence and reveals that both L11S and L13P mutants are markedly impaired with respect to cotranslational processing, accounting for the observed parathyroid dysfunction.

Adult↗

PubMed2004

Functional characterization of calcium-sensing receptor codon 227 mutations presenting as either familial (benign) hypocalciuric hypercalcemia or neonatal hyperparathyroidism.

Familial benign hypocalciuric hypercalcemia (FBHH), in which calcium homeostasis is disordered, can be distinguished from mild primary hyperparathyroidism by the finding of a heterozygous loss-of-function mutation in the calcium-sensing receptor (CaSR). Here, we report a Polish kindred with FBHH, the proband of which had undergone an unsuccessful parathyroidectomy. Direct sequence analysis of exon 4 of her CASR gene identified a heterozygous R227Q mutation in the extracellular domain of the receptor. This mutation segregated with other affected family members. A de novo heterozygous R227L mutation had previously been identified in a case of neonatal hyperparathyroidism. We performed a functional analysis by transiently transfecting wild-type and mutant (R227Q, R227L) CaSRs in human embryonic kidney (HEK293) cells. Both mutant receptors were expressed at a similar level to that of the wild-type, demonstrated a 160-kDa molecular species consistent with having undergone full maturation, and were visualized on the cell surface. Although both mutants were impaired in their MAPK responses to increasing extracellular calcium concentrations relative to wild type, this was more marked for R227L (EC(50) = 9.7 mM) than R227Q (EC(50) = 7.9 mM) relative to wild type (EC(50) = 3.7 mM). When cotransfected with wild-type CaSR to mimic the heterozygous state, the curves for both R227Q and R227L were right shifted intermediate to the curves for wild type and the respective mutant. This differential responsiveness may account, in part, for the markedly different clinical presentation of the R227Q mutation, classic FBHH, vs. the neonatal hyperparathyroidism of the R227L mutation.

Amino Acid Substitution↗

PubMed2004

CASRdb: calcium-sensing receptor locus-specific database for mutations causing familial (benign) hypocalciuric hypercalcemia, neonatal severe hyperparathyroidism, and autosomal dominant hypocalcemia.

Familial hypocalciuric hypercalcemia (FHH) is caused by heterozygous loss-of-function mutations in the calcium-sensing receptor (CASR), in which the lifelong hypercalcemia is generally asymptomatic. Homozygous loss-of-function CASR mutations manifest as neonatal severe hyperparathyroidism (NSHPT), a rare disorder characterized by extreme hypercalcemia and the bony changes of hyperparathyroidism, which occur in infancy. Activating mutations in the CASR gene have been identified in several families with autosomal dominant hypocalcemia (ADH), autosomal dominant hypoparathyroidism, or hypocalcemic hypercalciuria. Individuals with ADH may have mild hypocalcemia and relatively few symptoms. However, in some cases seizures can occur, especially in younger patients, and these often happen during febrile episodes due to intercurrent infection. Thus far, 112 naturally-occurring mutations in the human CASR gene have been reported, of which 80 are unique and 32 are recurrent. To better understand the mutations causing defects in the CASR gene and to define specific regions relevant for ligand-receptor interaction and other receptor functions, the data on mutations were collected and the information was centralized in the CASRdb (www.casrdb.mcgill.ca), which is easily and quickly accessible by search engines for retrieval of specific information. The information can be searched by mutation, genotype-phenotype, clinical data, in vitro analyses, and authors of publications describing the mutations. CASRdb is regularly updated for new mutations and it also provides a mutation submission form to ensure up-to-date information. The home page of this database provides links to different web pages that are relevant to the CASR, as well as disease clinical pages, sequence of the CASR gene exons, and position of mutations in the CASR. The CASRdb will help researchers to better understand and analyze the mutations, and aid in structure-function analyses.

Database Management Systems↗

PubMed2003

Recurrent familial hypocalcemia due to germline mosaicism for an activating mutation of the calcium-sensing receptor gene.

De novo activating mutations in the calcium-sensing receptor (CASR) gene are a common cause of sporadic isolated hypoparathyroidism. Here, we describe a family in which two affected siblings were found to be heterozygous for a novel F788L mutation in the fifth transmembrane domain encoded by exon 7 of the CASR. Both parents and the third sibling were clinically unaffected and genotypically normal by direct sequencing of their leukocyte exon 7 PCR amplicons. However, the mother was revealed to be a mosaic for the mutation by sequence analysis of multiple subclones as well as denaturing HPLC of the CASR exon 7 leukocyte PCR product. A functional analysis of the mutation was performed by transiently transfecting wild-type and mutant CASRs tagged with a c-Myc epitope in human embryonic kidney (HEK293) cells. The mutant CASR was expressed at a similar level as the wild type. The F788L mutant produced a significant shift to the left relative to the wild-type CASR in the MAPK response to increasing extracellular calcium concentrations. This is the first report of mosaicism for an activating CASR mutation and suggests that care should be exercised in counseling for risks of recurrence in a situation where a de novo mutation appears likely.

Adolescent↗