The goal of this grant proposal is to advance our understanding of the structure and pathophysiologic function of the human TSH receptor (TSHR). For this purpose, we will study:- 1. The role of the ectodomain in TSHR function. The TSHR ectodomain plays a paradoxical role in receptor activation. It constrains TSHR activity (removal of the ectodomain partially activates the TSHR). Conversely, the ectodomain is required for full TSHR activation by TSH. Partial TSHR activation by trypsin provides an important tool to understand the mechanism of ectodomain constraint and we will therefore study the mechanism underlying this phenomenon. To address the role of the ectodomain in full TSHR activation by ligand, we will mutagenize the TSHR to identify the region(s) in the ectodomain that lead to receptor activation following TSH binding. 2. TSHR ectodomain structure. Elucidation of the TSHR ectodomain structure will facilitate our primary goal of defining TSHR autoantibody binding sites. We have generated a CHO cell line that secretes large quantities of the TSHR ectodomain truncated at amino acid residue 289, corresponding approximately to the A subunit, the major site of autoantibody binding. By purifying mg quantities of TSHR-289 we will examine its disulfide bonds and glycan moieties. We will also generate large quantities of the full TSHR ectodomain (approximately 400 amino acids) to determine the sites of cleavage into A and B subunits, with the loss of a C peptide region. Finally, we will attempt to determine the 3- dimensional structure of TSHR-289 following partial deglycosylation of the molecule. 3. TSHR intermolecular interactions. We have found that expression of increasing numbers of TSHR on a cell leads to reduced TSH binding affinity and TSHR activation, or 'negative cooperativity', a process that appears to involve transient, non- covalent interactions between receptors. Our goal is to identify the interacting region(s) of the TSHR involved in the process. 4. TSHR antibodies and autoantibodies. We will generate mouse monoclonal antibodies (mAb) as tools to study autoantibody binding to the native, conformationally intact TSHR and, in other studies, we will study properties of the human autoantibodies themselves. MAb epitopes will be mapped using TSH-LH receptor chimeric receptors. The genes for the mAb will be cloned and used in H and L chain shuffling experiments. We will use purified TSHR-289 to affinity purify TSHR polyclonal or oligoclonal autoantibodies from patient serum. These studies will permit determination for the first time of the affinity of human autoantibodies for the TSHR. Finally, we will test the hypothesis that TSHR oligoclonality occurs because of early clinical presentation of Graves' disease. For this purpose, we will compare TSHR autoantibody k or lambda, L chain restriction in early and late disease.
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