Studies of FSH,FSH-analogues and the FSH-receptor

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DESCRIPTION (provided by applicant): Modification of the carbohydrate content of hFSH has significant effect on the physiologic properties of the hormone in vivo. To date, preliminary characterization of one such modification has been investigated: hFSH tethered to the CTP from the beta-subunit of hCG (either as a single-chain peptide or as a dimer associated with alpha-subunit). This modification increases the half-life and in vivo bioactivity of the hormone. The effect of fewer or greater numbers of O-linked carbohydrates, the impact on spacing of these moieties, and the potential role of N-linked sugars in this capacity have yet to be thoroughly investigated. These studies will generate important data as to general principles for carbohydrate physiology. By defining the role of differential glycosylation, therapeutic derivatives may be developed through carbohydrate manipulations that may be applicable to all the glycoprotein hormones as well as recombinant proteins in general. In addition, we have preliminary data indicating the feasibility of producing bifunctional hFSH analogues consisting of hFSH tethered to a growth factor. Bifunctionality, both in vitro and in vivo at the level of the ovary was confirmed. This allows for continued and expanded studies on the role of various targeted growth factors on ovarian physiology. Synergistic action on follicular development may also have therapeutic implications for women requiring ovarian stimulation for infertility. Structural knowledge of the interface between FSH and its receptor is essential to developing methods to perturb receptor activity. We plan to determine the structure of the extracellular domain (ECD) of the hFSH receptor, a member of the G protein- coupled receptor superfamily. A three-dimensional representation of the binding region of a glycoprotein hormone receptor will be elucidated for the first time. The structural dynamics and conformational changes in the receptor following ligand binding will provide data crucial to our understanding of the conditions requisite for receptor activation. Together, the studies described in this application will greatly enhance our understanding of gonadotropin biology, the role of various cofactors in ovarian physiology and elicit new methods for altering pharmacodynamics and pharmacokinetics of recombinant proteins.