LH, CG, FSH, and TSH constitute a family of structurally related glycoproteins which have major roles in physiology ranging from control of reproduction to control of thyroid function. Although nearly inactive themselves, the beta-subunits determine the activities of the intact hormones. The amino acid sequences of the beta-subunit have been elucidated for several hormones, however, the relationship between the structure and functions of these proteins is poorly understood. In this application, we propose to study the relationship between hCG beta- subunit structure and function by preparing beta-subunit analogs and studying their abilities to form dimers with alpha-subunit, to bind to receptors, and to be recognized by a panel of monoclonal antibodies. Our long-term goal will be to produce glycosylated derivatives of hCG which have low efficacy and which are partial agonists or antagonists. Our first specific aim will be to identify residues which are important for receptor binding and function. Taking advantage of the observations that hCG binds to rat receptors better than ovine LH (oLH) and that oLH binds to ram testes receptors much better than hCG, we will determine which residues of hCG beta-subunit can be replaced by residues of oLH beta-subunit to give an hCG based analog which will bind to ovine and rodent LH receptors like ovine LH. We will also be attempting to prepare glycosylated hCG analogs which have high affinity for rat tested LH receptors but which have low efficacy. We have already succeeded in making one such analog. In our second aim, we plan to determine the locations of residues which form binding sites for monoclonal antibodies. We have found that chimeras of hLH and hCG can be used to identify epitopes for a series of monoclonal antibodies and we will continue these studies to obtain greater resolution. Particular attention will be paid to identifying epitopes near the receptor binding domain which appear to be formed when the beta-subunit combines with alpha-subunit. We will combine the receptor binding data, the antibody binding data, and our existing epitope map to develop a model which explains changes in structure which occur when the beta-subunit combines with alpha-subunit and when the hormone binds to receptors. Our final specific aim will be to determine the assignment of three disputed disulfide bonds. We will make analogs which can be cleaved by CNBr and trypsin into disulfide linked fragments. The disulfide bonds can be assigned once the amino acid sequences of the fragments are determined.
