The overall goal of this project is to understand the endocrinology of the human glycoprotein hormones, follicle-stimulating hormone (FSH), choriogonadotropin (CG), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), and thereby to develop diagnostic and therapeutic applications for these hormones. Research advances in the current period include: First, we determined the roles of the FSH oligosaccharides. The alphaAsn52 oligosaccharide had a disproportionate role in signal transduction, but the other oligosaccharides had cooperative roles, so that as the number of oligosaccharides decreased, binding increased, and signal transduction decreased. As a result FSH lacking all its oligosaccharides behaved as a potent FSH antagonist. Second, we identified sites on FSH mediating receptor binding distinct from signal transduction. We substituted neutral or negatively charged residues for the positively charged Arg35 and Lys86 of betaFSH and found they were involved in receptor binding, but not signal transduction. Isolation of discrete foci for receptor binding and signal transduction forms the basis for the development of FSH agonists and antagonists. Third, we made mutations in the human FSH receptor (hFSH-R) gene to identify sites of the hFSH-R important for receptor binding and signal transduction. Arg or Asp substitutions at Ala571 in the third cytosolic loop of the seven transmembrane spanning domain increased receptor binding affinity 15 fold, while maintaining signal transduction. An Ile substitution constitutively activated adenylyl cyclase activity, analogous to the TSH receptor mutations found in patients with follicular adenomas. Fourth, we isolated an LH/CG receptor clone and demonstrated its functional expression in cos7 cells. Future directions of the project will emphasize the study of the structure-function characteristics of human FSH, LH, and CG and their homologous human receptors using side-directed mutagenesis of their respective genes.
