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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK019289-28
Application #
6623660
Study Section
Endocrinology Study Section (END)
Program Officer
Akolkar, Beena
Project Start
1976-05-01
Project End
2007-05-31
Budget Start
2003-07-01
Budget End
2004-05-31
Support Year
28
Fiscal Year
2003
Total Cost
$424,859
Indirect Cost
Name
Cedars-Sinai Medical Center
Department
Type
DUNS #
075307785
City
Los Angeles
State
CA
Country
United States
Zip Code
90048
McLachlan, Sandra M; Lesage, Sylvie; Collin, Roxanne et al. (2017) Genes Outside the Major Histocompatibility Complex Locus Are Linked to the Development of Thyroid Autoantibodies and Thyroiditis in NOD.H2h4 Mice. Endocrinology 158:702-713
Ludwig, Ralf J; Vanhoorelbeke, Karen; Leypoldt, Frank et al. (2017) Mechanisms of Autoantibody-Induced Pathology. Front Immunol 8:603
McLachlan, Sandra M; Aliesky, Holly; Banuelos, Bianca et al. (2017) Variable Effects of Dietary Selenium in Mice That Spontaneously Develop a Spectrum of Thyroid Autoantibodies. Endocrinology 158:3754-3764
Rapoport, Basil; Banuelos, Bianca; Aliesky, Holly A et al. (2016) Critical Differences between Induced and Spontaneous Mouse Models of Graves' Disease with Implications for Antigen-Specific Immunotherapy in Humans. J Immunol 197:4560-4568
Rapoport, Basil; McLachlan, Sandra M (2016) TSH Receptor Cleavage Into Subunits and Shedding of the A-Subunit; A Molecular and Clinical Perspective. Endocr Rev 37:114-34
Rapoport, Basil; McLachlan, Sandra M (2016) TSH Receptor Cleavage Into Subunits and Shedding of the A-Subunit; A Molecular and Clinical Perspective. Endocr Rev 2016:23-42
Chen, Chun-Rong; Salazar, Larry M; McLachlan, Sandra M et al. (2015) Deleting the Redundant TSH Receptor C-Peptide Region Permits Generation of the Conformationally Intact Extracellular Domain by Insect Cells. Endocrinology 156:2732-8
Rapoport, Basil; Aliesky, Holly A; Chen, Chun-Rong et al. (2015) Evidence that TSH Receptor A-Subunit Multimers, Not Monomers, Drive Antibody Affinity Maturation in Graves' Disease. J Clin Endocrinol Metab 100:E871-5
Chen, Chun-Rong; Hubbard, Paul A; Salazar, Larry M et al. (2015) Crystal structure of a TSH receptor monoclonal antibody: insight into Graves' disease pathogenesis. Mol Endocrinol 29:99-107
Rapoport, Basil; Aliesky, Holly A; Banuelos, Bianca et al. (2015) A unique mouse strain that develops spontaneous, iodine-accelerated, pathogenic antibodies to the human thyrotrophin receptor. J Immunol 194:4154-61

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