Maintenance of a stable internal environment within complex organisms requires specialized cells that sense changes in the extracellular concentration of specific ions (such as Ca2+). Although the molecular nature of such ion sensors is unknown, parathyroid cells possess a cell surface Ca(2+)-sensing mechanism that also recognizes trivalent and polyvalent cations (such as neomycin) and couples by changes in phosphoinositide turnover and cytosolic Ca2+ to regulation of parathyroid hormone secretion. The latter restores normocalcaemia by acting on kidney and bone. We now report the cloning of complementary DNA encoding an extracellular Ca(2+)-sensing receptor from bovine parathyroid with pharmacological and functional properties nearly identical to those of the native receptor. The novel approximately 120K receptor shares limited similarity with the metabotropic glutamate receptors and features a large extracellular domain, containing clusters of acidic amino-acid residues possibly involved in calcium binding, coupled to a seven-membrane-spanning domain like those in the G-protein-coupled receptor superfamily.

Gain-of-function mutations in the calcium-sensing receptor are associated with a familial syndrome of hypocalcemia with hypercalciuria that needs to be distinguished from hypoparathyroidism.

Mice lacking the calcium-sensing receptor (Casr) were created to examine the receptor's role in calcium homeostasis and to elucidate the mechanism by which inherited human Casr gene defects cause diseases. Casr+/- mice, analogous to humans with familial hypocalciuric hypercalcemia, had benign and modest elevations of serum calcium, magnesium and parathyroid hormone levels as well as hypocalciuria. In contrast, Casr-/- mice, like humans with neonatal severe hyperparathyroidism, had markedly elevated serum calcium and parathyroid hormone levels, parathyroid hyperplasia, bone abnormalities, retarded growth and premature death. Our findings suggest that Casr mutations cause these human disorders by reducing the number of functional receptor molecules on the cell surface.

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