Glycoprotein hormones regulate reproduction and development through interactions with receptors in ovarian, testicular, and thyroid tissues. Efforts to design hormone agonists and antagonists useful for treating infertility and hyperthyroidism would benefit from a molecular understanding of hormone-receptor interaction. The structure of human FSH bound to part of the extracellular domain of the human FSH receptor has been determined at 2.9A resolution, but the manner in which the hormone is docked with the receptor in the crystals is inconsistent with observations obtained using functional cell surface receptors. The crystals lack a portion of the full-length receptor that we term the signaling-specificity domain (SSD) because it participates in ligand binding and is needed for full efficacy. Indeed, the absence of SSD residues derived from exon 10 of the human LH receptor has been shown to cause male infertility. We have developed a structural model of the entire receptor, including the SSD. Most regions of our models were built by threading the sequences of the LH, FSH, and TSH receptors through structures of proteins that are widely thought to fold in similar fashions. Unfortunately, the SSD is not similar in sequence to the structures of known proteins. Following an extensive search of the structure databases, we found a template that could be used to model this domain and have incorporated it into models of all three classes of receptors. These models indicate how these receptors distinguish their ligands and suggest a simple mechanism of signal transduction. Studies in Aim 1 will enable us to test our model of the SSD and will employ a scanning mutagenesis technique that we developed during the current funding period. Studies in Aim 2 will enable us to create disulfide bonds between hCG and the LH receptor and between hFSH and the FSH receptor to test model-based predictions of ligand binding and ligand binding specificity. Studies in Aim 3 will employ homolog scanning mutagenesis to learn why the human LHR is activated by an hCG analog that is a potent antagonist in rat LHR assays. These will also enable us to test the role of the SSD in efficacy. Studies in Aim 4 will use total internal resonance fluorescence microcopy (TIRFM) to determine if the receptor exists on the cell surface as a homodimer. Other studies in this aim will determine if LH receptor analogs block the functions of the native LH (dominant negative inhibition) and if forced dimerization will promote receptor activation or inactivation. ? ? ?
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