Nearly 30 mutations have been identified to date in the coding region of the extracellular calcium-sensing receptor (CaR) that are associated with inherited human hypo-and hypercalcemic disorders. To understand the mechanisms by which the mutations alter the function of the receptor may help to discern the structurefunction relationships in terms of ligand-binding and G protein coupling. In the present studies, we transiently expressed eight known CaR mutations in HEK293 cells. The effects of the mutations on extracellular calciumand gadolinium-elicited increases in the cytosolic calcium concentration were then examined. Seven inactivating mutations, which cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism, show a reduced functional activity of the receptor because they may 1) reduce its affinity for agonists; 2) prevent conversion of the receptor from a putatively immature, high mannose form into the fully glycosylated and biologically active form of the CaR, in addition to lowering its affinity for agonists; or 3) fail to couple the receptor to and/or activate its respective G protein(s). Conversely, one activating mutation, which causes a form of autosomal dominant hypocalcemia, appears to increase the affinity of the receptor for its agonists.The recently cloned extracellular Ca 2ϩ (Ca 2ϩ o )-sensing receptor (CaR) 1 (1) has provided key insights into the pathogenesis of inherited human hypo-and hypercalcemic disorders (2, 3). The receptor, BoPCaR (bovine parathyroid Ca 2ϩ -sensing receptor) was first isolated from bovine parathyroid using expression cloning in Xenopus laevis oocytes and shows pharmacological properties nearly identical to those of the native receptor in its responses to extracellular divalent cations (i.e. Ca o ), and polyamines (e.g. neomycin) (1). Subsequently, cDNAs encoding the human homolog of the same receptor have been cloned from human parathyroid (4) and kidney (5), using a homology-based strategy. The human and bovine receptors share a high degree of homology at the amino acid level (93% identity). Stimulation of the CaR by agonists activates phospholipase C, with resultant increases in inositol phosphates and the cytosolic calcium concentration (Ca 2ϩ
The extracellular calcium (Ca 2؉ o )-sensing receptor (CaR) is a G protein-coupled receptor that plays important roles in calcium homeostasis. In this study, we employed epitope tagging, cell-surface biotinylation, and immunoprecipitation techniques to demonstrate that the CaR is expressed mostly in the form of a dimer on the surface of transfected human embryonic kidney (HEK293) cells. Western analysis of cell-surface proteins under nonreducing conditions showed that the CaR exists in several forms with molecular masses greater than 200 kDa. Most of these high molecular mass forms of the receptor could be converted to a single monomeric species at 160 kDa under reducing conditions. This result suggests that the CaR forms dimers or even higher oligomers on the cell surface through intermolecular disulfide bonds that are sensitive to reducing agents. Consistent with this hypothesis, use of a cell-surface crosslinking agent substantially increases the proportion of the putative dimeric CaR at 280 kDa relative to the monomeric form of the receptor at 160 kDa under reducing conditions. Dimerization of the CaR in intact cells was further demonstrated when we co-transfected and co-immunoprecipitated the wild type, full-length receptor and a truncated form of the CaR lacking its cytoplasmic tail. Taken together, we conclude from these results that the functional CaR resides on the cell surface of transfected HEK293 cells in the form of a dimer.
We characterized the in vivo, cellular and molecular pathophysiology of a case of neonatal hyperparathyroidism (NHPT) resulting from a de novo, heterozygous missense mutation in the gene for the extracellular Ca 2 ϩ ( )-sensing receptor (CaR). The female neonate presented with moderately severe hypercalcemia, markedly undermineralized bones, and multiple metaphyseal fractures. Subtotal parathyroidectomy was performed at 6 wk; hypercalcemia recurred rapidly but the bone disease improved gradually with reversion to an asymptomatic state resembling familial benign hypocalciuric hypercalcemia (FBHH). Dispersed parathyroid cells from the resected tissue showed a set-point (the level of half maximally inhibiting PTH secretion) substantially higher than for normal human parathyroid cells ( ف 1.8 vs.
We recently demonstrated that the G protein-coupled, extracellular calcium-sensing receptor (CaR) forms disulfide-linked dimers. The functional significance of dimerization of this receptor was suggested by our earlier observations that CaRs carrying certain point mutations exert dominant negative effects on the function of the coexpressed wild-type receptor both in vivo and when cotransfected in human embryonic kidney cells. In this study, we explored the functional consequences of CaR dimerization. Coexpression in human embryonic kidney cells of specific pairs of mutant CaRs, each with reduced or absent activity because of distinct loss-of-function mutations, results in the formation of heterodimers and partially reconstitutes extracellular calcium-dependent signaling. Moreover, our results suggest that the CaR has at least two functionally separable domains. However, the presence of an abnormal domain in each mutant monomer substantially impairs the function of the CaR heterodimer, resulting in the reconstituted CaRs having characteristics distinct from those of the wild-type CaR. Our study suggests that intermolecular interactions within the dimeric CaR are important for the receptor's function. The extracellular calcium ([Ca2ϩ
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