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
AUTOR(ES)
Chen, Chun-Rong
FONTE
The Endocrine Society
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2703505Documentos Relacionados
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