The overall objective of this project continues to be the achievement of an understanding at the molecular level, of the interaction of FSH with its receptor. Information obtained during the previous period of grant support will be utilized for development of a procedure of isolation of purified and stable FSH binding protein/receptors. Experiments will be undertaken to distinguish between the FSH binding protein and a functional receptor after reconstitution of the putative receptor into phospholipid vesicles and fusion with turkey erythrocyte membranes. The endpoint will be measurement of adenyl cyclase stimulation in response to FSH binding. The purified receptor and its putative subunits will be characterized in terms of physical properties (molecular weight, shape, isoelectric point, etc.) and chemical composition. Limited sequence analysis will be attempted in order to further characterize the subunits and form the basis for construction of cDNA probes. We will attempt to develop an FSH sensitive receptor-G/protein-adenyl cyclase system reconstituted in phospholipid vesicles of defined composition in order to allow detailed studies of the molecular interactions involved. Polyclonal and monoclonal antibodies will be generated to the receptor and/or its subunits and will be utilized for development of an assay for the receptor to be utilized in physiologic studies or as structural probes. The role of the carbohydrate component of FSH in receptor binding and initiation of post binding events will also be examined. In particular, we will search for the possible existence of a membrane lectin specifically associated with the FSH stimulated adenyl cyclase response. A variety of related studies are described which will be undertaken as circumstances allow.
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