Endocrinology, Vol 136, 521-527, Copyright © 1995 by Endocrine Society

ARTICLES

Folding of the recombinant human thyrotropin (TSH) receptor extracellular domain: identification of folded monomeric and tetrameric complexes that bind TSH receptor autoantibodies

PN Graves, H Vlase and TF Davies
Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029.

We have analyzed protein folding and disulfide bond formation in the extracellular domain of the human TSH receptor (hTSHR-ecd) expressed in Escherichia coli. This domain, which begins at the amino-terminus and ends at residue 415, is a major autoantigen in human autoimmune thyroid disease. Refolding of reduced and denatured hTSHR-ecd occurred in polyacrylamide gels treated with 0.25 M KCl for visualization of protein bands. Under conditions of partial renaturation, at least three forms of the hTSHR-ecd were resolved by reelectrophoresis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis: 1) unfolded monomers, 2) folded monomers, and 3) tetramers. Disulfide bond formation was implicated in both folding and tetramerization, as reduction of these forms produced only unfolded monomers. A natural variant of the hTSHR (v1.3), sharing 231 N-terminal amino acids with hTSHR-ecd, formed folded monomers and dimers on renaturation, but not tetramers, implicating one or more of the five cysteine residues residing between positions 231-415 in the association of dimers into tetramers. Binding of three different sources of hTSHR antibodies to these various forms of the hTSHR-ecd was assessed by immunoblotting using: 1) murine monoclonal antibodies (MAbs) generated against hTSHR- ecd, 2) rabbit polyclonal antisera generated against overlapping synthetic peptides spanning residues 37-71 of the hTSHR-ecd, and 3) human immunoglobulin G from patients with Graves' disease and detectable hTSHR-Ab. One of the MAbs, shown to recognize residues 21- 35, and the rabbit polyclonal antibodies bound to all three forms of the hTSHR-ecd. Some of the hTSHR autoantibodies bound predominantly to the monomeric forms of the hTSHR, but autoantibodies were also identified that recognized tetrameric hTSHR-ecd. These data demonstrate that hTSHR-Abs recognize differing nonlinear and linear epitopes in the hTSHR-ecd and provide a methodology that should be useful for further defining the structural requirements for folding of this functionally and immunologically important domain of the hTSHR.

This article has been cited by other articles:

				I. Ciullo, R. Latif, P. Graves, and T. F. Davies Functional Assessment of the Thyrotropin Receptor-{beta} Subunit Endocrinology, July 1, 2003; 144(7): 3176 - 3181. [Abstract] [Full Text] [PDF]

				R. Latif, P. Graves, and T. F. Davies Ligand-dependent Inhibition of Oligomerization at the Human Thyrotropin Receptor J. Biol. Chem., November 15, 2002; 277(47): 45059 - 45067. [Abstract] [Full Text] [PDF]

				M. H. Lee, J. Y. Park, B. Y. Cho, and C.-B. Chae Expression of the Functional Extracellular Domain of Human Thyrotropin Receptor Using a Vaccinia Virus System: Its Purification and Analysis of Autoantibody Binding J. Clin. Endocrinol. Metab., April 1, 1999; 84(4): 1391 - 1397. [Abstract] [Full Text]

				B. Rapoport, G. D. Chazenbalk, J. C. Jaume, and S. M. McLachlan The Thyrotropin (TSH)-Releasing Hormone Receptor: Interaction with TSH and Autoantibodies Endocr. Rev., December 1, 1998; 19(6): 673 - 716. [Abstract] [Full Text]

				S. Costagliola, P. Rodien, M.-C. Many, M. Ludgate, and G. Vassart Genetic Immunization Against the Human Thyrotropin Receptor Causes Thyroiditis and Allows Production of Monoclonal Antibodies Recognizing the Native Receptor J. Immunol., February 1, 1998; 160(3): 1458 - 1465. [Abstract] [Full Text] [PDF]

				G. D. Chazenbalk, J. C. Jaume, S. M. McLachlan, and B. Rapoport Engineering the Human Thyrotropin Receptor Ectodomain from a Non-secreted Form to a Secreted, Highly Immunoreactive Glycoprotein That Neutralizes Autoantibodies in Graves' Patients' Sera J. Biol. Chem., July 25, 1997; 272(30): 18959 - 18965. [Abstract] [Full Text] [PDF]

				A. Kakinuma, G. D. Chazenbalk, J. C. Jaume, B. Rapoport, and S. M. McLachlan The Human Thyrotropin (TSH) Receptor in a TSH Binding Inhibition Assay for TSH Receptor Autoantibodies J. Clin. Endocrinol. Metab., July 1, 1997; 82(7): 2129 - 2134. [Abstract] [Full Text] [PDF]

				H. Vlase, N. Matsuoka, P. N. Graves, R. P. Magnusson, and T. F. Davies Folding-Dependent Binding of Thyrotropin (TSH) and TSH Receptor Autoantibodies to the Murine TSH Receptor Ectodomain Endocrinology, April 1, 1997; 138(4): 1658 - 1666. [Abstract] [Full Text] [PDF]

				J. C. Jaume, A. Kakinuma, G. D. Chazenbalk, B. Rapoport, and S. M. McLachlan Thyrotropin Receptor Autoantibodies in Serum Are Present at Much Lower Levels Than Thyroid Peroxidase Autoantibodies: Analysis by Flow Cytometry J. Clin. Endocrinol. Metab., February 1, 1997; 82(2): 500 - 507. [Abstract] [Full Text] [PDF]

				Y. Bobovnikova, P. N. Graves, H. Vlase, and T. F. Davies Characterization of Soluble, Disulfide Bond-Stabilized, Prokaryotically Expressed Human Thyrotropin Receptor Ectodomain Endocrinology, February 1, 1997; 138(2): 588 - 593. [Abstract] [Full Text] [PDF]

				J. Y. Park, I. J. Kim, M. H. Lee, J. K. Seo, P. G. Suh, B. Y. Cho, S. H. Ryu, and C.-B. Chae Identification of the Peptides That Inhibit the Stimulation of Thyrotropin Receptor by Graves' Immunoglobulin G from Peptide Libraries Endocrinology, February 1, 1997; 138(2): 617 - 626. [Abstract] [Full Text] [PDF]

				R. Latif, P. Graves, and T. F. Davies Oligomerization of the Human Thyrotropin Receptor. FLUORESCENT PROTEIN-TAGGED hTSHR REVEALS POST-TRANSLATIONAL COMPLEXES J. Biol. Chem., November 21, 2001; 276(48): 45217 - 45224. [Abstract] [Full Text] [PDF]