The long-range goals of this project include elucidation of the conformational and structure-function relationships of the placental-derived glycoprotein hormone, human chorionic gonadotropin (hCG), delineation of the various intracellular signaling pathways, and characterization of relatively unexplored cellular responses to hCG. A working model has been developed for CG and its pituitary-derived counterpart, luteinizing hormone (LH), that combines the results of chemical and enzymatic modifications, immunological mapping, and physicochemical studies. In order to experimentally test this model, site-directed mutagenesis and trace labeling studies have been designed to elucidate the subunit contact sites for heterodimer formation and the amino acid sequence determinants for receptor binding and activation. Numerous studies have emphasized the role of cAMP in gonadotropin-mediated intracellular signaling, and we also have tentative evidence for the involvement of inositol-1,4,5 trisphosphate and possibly other inositol-- containing phospholipids; this hypothesis of alternate signaling will be experimentally tested in transformed Leydig cells. This project will also delineate some of the cellular responses to gonadotropin and mutant proteins. These include a dramatic CG/LH-mediated morphological change in transformed Leydig cells (MA-10), which we propose is due to cytoskeletal reorganization; a proposed CG/LH-regulated interaction of cells with the extracellular matrix, involving such modulators as SPARC (Secreted Protein that is Acidic and Rich in Cysteine); and CG/LH regulation of the oncogenes c-fos, c-jun, and c-myc. This. comprehensive project is designed to elucidate amino acid sequence determinants regulating heterodimer formation and receptor recognition/activation, intracellular signaling pathways possibly distinct from cAMP, and relatively unexplored aspects of hCG function.
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