HCG STRUCTURE/FUNCTION RELATIONSHIPS

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The long-range goal of this project is elucidation at the molecular level of the structural and functional aspects of the two glycoprotein hormones, hCG and LH, and their common G protein-coupled receptor, LH/CGr. This research is guided by the hypotheses that (i) discrete regions of the alpha and beta subunits may function both in holoprotein formation and receptor binding, and (ii) conformation changes occur upon heterodimer formation and receptor binding. The work will be a continuation of current studies designed to identify and delineate amino acid residues and discrete regions of the alpha and beta subunits that are important in hormone function. Site-directed mutagenesis will be used to replace or delete certain amino acid residues and putative functional domains that are known or suspected to be involved in secretion, subunit assembly, receptor binding, and perhaps both subunit and receptor binding. A number of the beta replacements will be made in the Keutmann steroidogenic loop (residues 38-57), the Ward determinant loop (residues 93-lOO), and other regions identified as important in function. These mutant forms of hCG will be assayed in vitro via competitive binding and steroidogenesis. Differential trace labeling will be done with [3H]acetic anhydride to map the reactivities of amino groups in ovine LH and equine CG for comparison with hCG. A selected number of residues on LH/CGr will be replaced by site-directed mutagenesis and the mutants assayed for hormone binding and cAMP stimulation. These results will provide information on the roles of specific groups of the extracellular domain and extracellular connecting loops on hormone binding and the function of residues in the transmembrane helices and intercellular connecting loops on signal transduction. Physicochemical studies will be conducted on a few of the more interesting C-terminal deletion mutants, e.g. those where stable tertiary structure is forming, to gather data on their conformational aspects. The gonadotropins hCG and LH, acting through their common receptor, are required for gonadal steroidogenesis, ovulation, and maintenance of early pregnancy. Elucidation of their structure-function relationships may provide new insights into fertility regulation and the etiology of infertility.