Brown and colleagues (Nature 1993) have cloned a novel calcium-sensing receptor (CaR) which is a member of the G protein-coupled receptor superfamily similar in structure to the metabotropic glutamate receptors. The CaR is expressed in a limited range of cell types including kidney, brain, thyroid C cells, and most prominently parathyroid cells. The CaR cDNA predicts a 7 transmembrane core typical of G protein-coupled receptors but with a large (approximately 600 residue) N-terminal extracellular domain (ECD). We are studying several aspects of the receptor's structure and function in order to understand how calcium binding to the receptor leads to G protein activation. We have raised polyclonal antisera to several synthetic peptides corresponding to sequences in the large extracellular domain of the receptor. These recognize the receptor on western blots. Monoclonal antibodies were raised against the 2 most immunogenic ECD peptides. These monoclonals have been extensively characterized and have proved very useful in immunoblot, immunocytochemistry, and flow cytometry studies of the receptor. For example we have been able to show that glycosylation of the ECD is essential for CaR expression at the cell surface. We have also defined by mutagenesis, regions of the 200 residue C-terminus critical for receptor expression and G protein coupling. Detailed mutagenesis studies of key residues (e.g. conserved cysteines, putative glycosylation sites) in the ECD have also been performed, and have defined cysteines and glycosylation sites critical for receptor expression at the cell membrane. We have succeeded in expressing and purifying the ECD. 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. We have also succeeded in generating a battery of monoclonal antibodies against the purified ECD which have interesting functional effects on the CaR, and which are being evaluated for their epitopes in a further effort to define receptor structure/function. Finally, 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.
