title: Insight into thyroid-stimulating autoantibody interaction with the thyrotropin receptor N-terminus based on mutagenesis and re-evaluation of ambiguity in this region of the receptor crystal structure.
abstract: BACKGROUND
  Thyroid-stimulating autoantibodies (TSAb) bind to the thyrotropin receptor (TSHR) extracellular domain, or ectodomain (ECD), comprising a leucine-rich repeat domain (LRD) linked by a hinge region to the transmembrane domain (TMD). The LRD (residues 22-260; signal peptide 1-21) contains two disulfide-bonded loops at its N-terminus. In the crystal structure of the isolated LRD complexed with human TSAb monoclonal antibody (mAb) M22, N-terminal disulfide loop 1 (residues 22-30) could not be determined because of crystal disorder. Nevertheless, present crystal structure data are interpreted to exclude a role for the LRD N-terminal disulfide loops in the TSAb epitope(s), contradicting prior functional evidence of a role for these loops in TSAb function.
  
  
  METHODS
  To re-examine this issue we studied two cell types expressing the TSHR with the extreme N-terminal loop 1 (residues 22-30) deleted: the TSHR ECD lacking the TMD and tethered to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) anchor, and the TSH holoreceptor containing the TMD. Because TSAb including M22 "see" the holoreceptor poorly relative to the TSHR ECD-GPI, we used the latter to examine the effect of deleting residues 22-30 on M22 binding by flow cytometry and the holoreceptor to test the effect of this deletion on the functional response to M22.
  
  
  RESULTS
  Deletion of TSHR N-terminal loop 1 (residues 22-30) reduced the number of TSHR-ECD-GPI recognized by M22 relative to two TSHR mAb with epitopes far downstream of the LRD N-terminal loops. Relative to control mAb 2C11, M22 recognized only 60.4% of cell surface receptors (p = 0.02). In contrast to M22 binding to TSHR-ECD-GPI, in functional studies with the TSH holoreceptor, M22 stimulation of cAMP generation was unaltered by the loop 1 deletion.
  
  
  CONCLUSIONS
  Our data support the concept that TSAb interact with the cysteine-rich N-terminus of the TSHR. Comparison of crystal structures of the same TSHR LRD in complex with TSAb M22 or blocking antibody K1-70 helps reconcile contradictory viewpoints. A difference between M22 interaction with the identical TSHR N-terminus expressed on the TSHR-ECD-GPI and holoreceptor suggests that crystallization of the TSHR LRD-M22 complex may not provide a complete understanding of the functional TSAb epitope(s) in Graves' disease.
authors.affiliation: Thyroid Autoimmune Disease Unit, Cedars-Sinai Research Institute, Los Angeles, California 90048, USA.
authors.forename: Sepehr
authors.initials: S
authors.lastname: Hamidi
authors.forename: Chun-Rong
authors.initials: CR
authors.lastname: Chen
authors.forename: Sandra M
authors.initials: SM
authors.lastname: McLachlan
authors.forename: Basil
authors.initials: B
authors.lastname: Rapoport
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000378:metabolism; D001666:Binding Sites, Antibody; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003461:Crystallography; D018604:Epitope Mapping; D000939:Epitopes; D005434:Flow Cytometry; D018828:Immunoglobulins, Thyroid-Stimulating / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D054730:Protein Interaction Domains and Motifs; D011989:Receptors, Thyrotropin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D018828:Immunoglobulins, Thyroid-Stimulating; D011989:Receptors, Thyrotropin