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 prtoein coupling. Detailed mutagenesis studies of key residues (e.g. conserved cysteines, putative glycosylation sites) in the ECD are also being performed.We have entered into a CRADA with the long-range goal of generating monoclonal antibodies against the ECD for use as imaging reagents for parathyroid tumors in patients with failed neck explorations. In the course of this work we have succeeded in expressing, purifying, and biochemically characterizing the ECD. 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 utility in binding to parathyroid tissue.