Brown and colleagues (Nature 1993) have cloned a novel calcium-sensing receptor (CaR) which is a member of the G protein-coupled receptor (GPCR) superfamily, specifically subfamily 3 which includes metabotropic glutamate, GABA-B, taste, and putative pheromone receptors. The CaR is expressed in a variety of cell types including kidney, brain, thyroid C cells, and most prominently parathyroid cells, and is involved in extracellular calcium homeostasis. The CaR cDNA predicts a 7 transmembrane core typical of GPCR but with a large (approximately 600 residue) N-terminal extracellular domain (ECD). We are studying the receptors structure and function in order to understand how calcium binding to the receptor leads to G protein activation. We have raised polyclonal and monoclonal antibodies to synthetic peptides corresponding to sequences in the ECD of the receptor. These antibodies have proved very useful in immunoblot, immunocytochemistry, and flow cytometry studies of the receptor. We have alsosucceeded in expressing and purifying the ECD,and in generating monoclonal antibodies against the purified ECD. These have interesting functional effects on the CaR, and are being evaluated for their epitopes to help define receptor structure/function. Biochemical characterization of the ECD included N-terminal sequencing to define site of signal peptide cleavage, definition of carbohydrate content, secondary structure by CD, and sites of tryptic cleavage. We found that the ECD is an intermolecular disulfide-linked dimer that accounts for the dimeric nature of the intact receptor. Mutagenesis of ECD cysteines has defined which are essential for receptor expression and has identified the cysteine responsible for receptor dimerization. Disulfide mapping of the ECD is in progress.We have also identified the glycosylation sites critical for receptor expression at the cell surface. We have characterized the functional effects of missense mutations identified in subjects with autosomal dominant hypocalcemia. Most such mutations cause increased sensitivity of the receptor to calcium, but one in the 7th transmembrane domain causes true constitutive activation of the receptor, even in the context of a truncated receptor lacking the ECD.We have modeled the ECD structure based on its homology to bacterial periplasmic binding proteins known to have a bilobed, venus flytrap structure, and are testing this model using mutagenesis and biochemical approaches. - G protein-coupled receptor; hypercalcemia; hypocalcemia; parathyroid hormone; calcium homeostasis