CLONING OF THE PARATHYROID HORMONE RECEPTOR CDNA

甲状旁腺激素（PTH）在调节骨骼和神经系统中起着核心作用

Parathyroid hormone (PTH) has a central role in regulating bone and mineral metabolism, yet little is known of the mechanisms underlying PTH action at its receptor. My overall goal is to determine the molecular basis of PTH receptor action. The first aim of this proposal is to extend our biochemical characterization of the receptor, by defining the functional significance of a major proteolytic cleavage site that generates an apparently uncoupled receptor, and by better defining the role of glycosylation in receptor function. Our major objective, however, is the isolation of the PTH receptor cDNA. This will enable us to deduce its amino acid sequence, which in turn will provide a logical framework for studies designed to elucidate the structural basis of PTH receptor function. Two complementary cloning strategies will be used. First, we will use recently developed PTH receptor monoclonal antibodies to immunoscreen renal and bone cell cDNA libraries cloned in a lambda expression vector. A second approach will be to use the polymerase chain reaction (PCR) and degenerate oligonucleotide primers corresponding to highly conserved domains in closely related G(s)-coupled receptors to selectively amplify the PTH receptor cDNA from bone and kidney cDNA. Clones with the appropriate nucleotide and deduced amino acid sequence will be used to rescreen the original cDNA libraries to obtain near full-length clones, from which the entire PTH receptor sequence may be deduced. Once this is done we will validate the cloned sequence by eukaryotic expression of the full-length clones and assessment of specific 125I-bPTH(1-34) binding and PTH responsive adenylate cyclase activity. Completion of these aims will permit the future elucidation of the structural basis for PTH receptor function, using sitedirected mutagenesis and the construction of receptor chimeras to determine the specific domains responsible for ligand binding and G(s)-coupling. Successful completion of these studies will provide new insights into the molecular basis of function not only for the PTH receptor, but also for the family of G-protein coupled receptors.