Title : 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
Abstract :
- 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