The primary objective of this grant proposal is to further our understanding of the TSH receptor, in terms of its physiological interaction with TSH and its pathophysiological role in autoimmune thyroid disease. The following studies will be performed: 1. Studies on TSH binding to the TSH receptor: The binding of TSH to recombinant human TSH receptor variants will be used as a means to understand the structure of the receptor, in particular to help localize the specific regions that interact with TSH. Approaches to be used include the use of: a) TSH-LH/CG receptor chimeras, and b) individual amino acid substitutions. 2. TSH receptor signal transduction: TSH-LH/CG receptor chimeras as well as other TSH receptor mutants will be used to identify the extracellular domains and cytoplasmic regions of the TSH receptor that are involved in signal transduction. The role of these regions in both the CAMP and phosphatidyl inositol pathways will be studied. A possible role for phosphorylation in TSH receptor function will be investigated. 3. Overexpression and crystallization of the TSH receptor: Large quantities of recombinant TSH receptor protein are a requirement for crystallization and determination of the 3-dimensional structure of the molecule. Purified TSH receptor protein will also be invaluable for the generation of mouse and human TSH receptor monoclonal antibodies. Two approaches will be undertaken to attain this goal; namely dihydrofolate reductase (DHFR)-linked amplification and baculovirus expression. 4. Study of TSH receptor antibodies: The primary goal will be to define the epitopes on the TSH receptor that are recognized by TSH receptor antibodies in the sera of patients with autoimmune thyroid disease. Secondary goals will be the generation of monoclonal antibodies for TSH receptor purification. The approaches to be taken include: a) Screening of a TSH receptor CDNA fragment expression library with sera from patients with autoimmune thyroid disease; b) Generation of mouse anti-TSH receptor monoclonal antibodies; c) Generation of human anti-TSH receptor monoclonal antibodies.
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