The objective of the proposed study is to understand the correlations between structure and function of human gonadotropins and their receptors with respect to ovarian and testicular physiology. This knowledge is fundamental in the development of contraceptive agents and hormone antagonists, regulation of human fertility, and the diagnosis and treatment of human disease. In the proposed project, the use of synthetic peptides and recombinant receptor proteins will be employed to correlate specific regions of amino acid sequence with the functional activities of human gonadotropins and the LH/CG receptor.
The specific aims of this study are to: 1) delineate the effector/receptor binding sites (regions alpha1-15, alpha26-46 and alpha71-92) on the surface of the common alpha-subunit of the human gonadotropins LH and CG, and to identify the essential contact residues in the delineated regions that are involved in receptor binding; 2) develop synthetic hormone analogs (agonists and antagonists) with increased potency based upon the findings in Aims 1; 3) map the epitope regions of the beta-subunits of human LH, hCG, and hFSH by immunochemical methods with a series of synthetic hexapeptides and produce gonadotropin specific antibodies using synthetic peptide vaccines; 4) delineate specific amino acid sequences of the LH/CG receptor that participate in hormone binding and activation of adenylate cyclase by a comprehensive synthetic approach and correlate the hormone binding regions on the receptor with the receptor binding regions on the hormone; 5) develop a repertoire of polyclonal and monoclonal antibodies to synthetic receptor peptides and recombinant LH/CG receptor proteins. Anti-receptor antibodies will then be used for the quantitation, characterization, and immunohistochemical localization of receptor. Methods to be employed in these studies are: solid-phase peptide synthesis; amino acid analysis; gas-phase microsequencing; reversed-phase HPLC; ion-exchange and liquid chromatography; affinity chromatography; competitive radioreceptor binding assays; enzyme-linked immunosorbent assays; Leydig cell bioassays; hexapeptide pin-binding assays; hormone/receptor purification; oligonucleotide synthesis; primer directed dideoxy DNA sequencing; recombinant DNA cloning and baculovirus expression systems.
