A monoclonal antibody with thyrotropin (TSH) receptor inverse agonist and TSH antagonist activities binds to the receptor hinge region as well as to the leucine-rich domain.
Monoclonal antibody CS-17 is a TSH receptor (TSHR) inverse agonist (suppresses constitutive activity) and a TSH antagonist. Elucidation of the CS-17 epitope will provide insight into TSHR structure and function. Present information on its epitope conflicts with recent data regarding another TSHR inverse agonist antibody. To characterize further the CS-17 epitope, we exploited the observation that CS-17 does not recognize a chimeric receptor with TSHR hinge region residues 261-289 replaced with homologous rat LH receptor residues (13 mismatches). We generated individual and double TSHR mutations corresponding to these mismatches. On flow cytometry, only T273L/R274V reduced CS-17 recognition. No mutation affected TSH-stimulated cAMP generation. Because the immunogen for CS-17 generation was highly glycosylated, we also investigated whether the glycan moiety at N198, topologically adjacent to Y195 (a previously identified epitopic component), could contribute to the CS-17 epitope. Elimination of this N-linked glycan (mutations of N198 and T200) abrogated CS-17 binding without altering TSH responsiveness. However, studies with tunicamycin suggested that these mutations affected CS-17 binding by altering the polypeptide backbone rather than eliminating the glycan moiety. TSHR residues N198 and T200, like Y195, are on the convex facet of the leucine-rich domain. In summary, the present data indicate that the discontinuous epitope of CS-17, a TSHR inverse agonist and TSH antagonist, includes a component in the hinge region as well as the convex surface of the TSHR leucine-rich domain. These findings expand our present concept of glycoprotein hormone binding and function.
Chen
Chun-Rong
CR
Autoimmune Disease Unit, Cedars-Sinai Research Institute and University of California Los Angeles School of Medicine, Los Angeles, California 90048, USA.
McLachlan
Sandra M
SM
Rapoport
Basil
B
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000939:Epitopes / Q000235:genetics*; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D011134:Polysaccharides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011989:Receptors, Thyrotropin / Q000819:agonists* / Q000737:chemistry / Q000276:immunology; D013972:Thyrotropin / Q000037:antagonists & inhibitors*; D014415:Tunicamycin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011134:Polysaccharides; D011989:Receptors, Thyrotropin; D014415:Tunicamycin; D013972:Thyrotropin