The parathyroid (PTH) is the primary peptide regulating blood calcium in humans and other vertebrates. Understanding its mechanism of action is central to development of new treatments for metabolic abnormalities in the calcium/phosphate homeostatic system. Recently the emphasis in peptide hormone research has turned from study of the hormones to study of their cellular receptors. This proposal centers on the study of putative PTH receptors. Two such proteins have been purified to homogeneity and it is now possible to develop the tools of immunoaffinity chromatography and cDNA cloning for the further study of their structure and biological actions. The work has two major objectives: (a) the development of immunochromatographic methods for purification of larger amounts of these proteins and (b) the development of the tools of molecular biology for study of the structure of these proteins as their genes. It is proposed to utilize CNBr fragmentation of receptors and to obtain as much of its primary structure as possible using gas phase microsequencing methods. When this is achieved, larger amounts of the peptides will be synthesized and, in addition, oligonucleotide sequences which reflect these peptides will be prepared. The peptides will be used for immunizations with the goal of obtaining antisera the receptor proteins. These antibodies will be purified and used to prepare immunoaffinity columns for receptors. This will lead to the possibility of purification of the larger amounts of receptor needed for biochemical and biological studies. The oligonucleotides will be used for screening a bovine kidney cDNA expression library. With both oligonucleotide and antibody probes, we will have the tools required for isolation of the clones which contain receptor cDNAs. This will allow us to ultimately obtain the complete primary structure of the receptor mRNA (and thus the protein). Finally, with the use of the cDNAs and the oligonucleotide probes, we will be able to screen bovine genomic libraries for receptors and to begin work on determining the find structure of this gene.
