title: Identification of key amino acid residues in a thyrotropin receptor monoclonal antibody epitope provides insight into its inverse agonist and antagonist properties.
abstract: CS-17 is a murine monoclonal antibody to the human TSH receptor (TSHR) with both inverse agonist and antagonist properties. Thus, in the absence of ligand, CS-17 reduces constitutive TSHR cAMP generation and also competes for TSH binding to the receptor. The present data indicate that for both of these functions, the monovalent CS-17 Fab (50 kDa) behaves identically to the intact, divalent IgG molecule (150 kDa). The surprising observation that CS-17 competes for TSH binding to the human but not porcine TSHR enabled identification of a number of amino acids in its epitope. Replacement of only three human TSHR residues (Y195, Q235, and S243) with the homologous porcine TSHR residues totally abolishes CS-17 binding as detected by flow cytometry. TSH binding is unaffected. Of these residues, Y195 is most important, with Q235 and S243 contributing to CS-17 binding to a much lesser degree. The functional effects of CS-17 IgG and Fab on constitutive cAMP generation by porcinized human TSHR confirm the CS-17 binding data. The location of TSHR amino acid residues Y195, Q235, and S243 deduced from the crystal structure of the FSH receptor leucine-rich domain provides valuable insight into the CS-17 and TSH binding sites. Whereas hormone ligands bind primarily to the concave surface of the leucine-rich domains, a major portion of the CS-17 epitope lies on the opposite convex surface with a minor component in close proximity to known TSH binding residues.
authors.affiliation: Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048, USA.
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; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000494:pharmacology; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D005434:Flow Cytometry; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011989:Receptors, Thyrotropin / Q000819:agonists / Q000037:antagonists & inhibitors / Q000276:immunology*; D013552:Swine
chemical_list: D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D011989:Receptors, Thyrotropin; D000242:Cyclic AMP