The present proposal extends previous biochemical and immunochemical work aimed at understanding the FSH/FSH-receptor interaction. Data collected during the previous grant period has identified specific regions of both hFSH subunits (hFSHalpha, hFSHbeta) which by these methods are involved in FSH-receptor binding and FSH-subunit contact. The first objective of the present proposal is to prepare mutant hFSH which has been substituted with alanine at residues within the sequences identified as putative receptor binding sites. The operating hypothesis is that amino acids within sequence hFSHbeta - (33-53) and hFSHalpha- (73-92) are in part, the assembled receptor binding site of hFSH. Mutant hFSH will be tested to determine if the mutation causes an alteration in receptor binding and activation. The second objective is to mutate hFSH within sequences which have been identified as subunit association sites. Data collected during the previous grant period have identified sequences hFSHbeta- (81- 100) and hFSHalpha- (33-58) as containing amino acids which participate n subunit association to form heterodimeric hFSH. The hypothesis is that mutation of amino acids involved in subunit association will result in altered ability of subunits to form heterodimeric hFSH. Whether or not the mutated hFSH subunits will form heterodimers will be assessed. The third objective is corollary to the first two objectives. The applicant proposes to define the extracellular FSH binding domain of the FSH receptor. Recombinant rat FSH receptor will then be used to immunize mice to produce antibodies against the receptor. Monoclonal anti-receptor antibodies which block binding of hFSH to FSH-receptor will then be epitope mapped using FSH-receptor synthetic peptides. This will allow determination of specific residues which comprise in part the FSH binding site of the FSH-receptor. The long term goals are to understand the molecular (structural) basis of how human follicle stimulating hormone (hFSH) interacts with its receptor and how FSH subunits associate to form biologically active heterodimer. This understanding will contribute to the knowledge necessary to an FSH or FSH-receptor based, contraceptive vaccine. Additionally, perhaps a second generation of hormones that are more potent (agonistic) than hFSH or can inhibit (antagonist) hFSH action in vivo could be developed.
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