Abstract
Objectives: The human TSHR hinge region links the extracellular LRR domain with the transmembrane domain. Previously published crystal structures of a part of the extracellular TSHR or FSHR domain lack structural data for the hinge region. Recently, we identified positions E297, E303, and D382 in the hinge region, which are involved in bovine TSH (bTSH) binding. The negative charge of positions E297 and D382 is obviously essential in the process of bTSH binding, suggesting a complementary charge-charge interaction with positively charged residues of the hormone. Furthermore, the triple hinge mutant E297A/E303A/D382A showed a strongly reduced cAMP signaling effect after treatment with bTSH.
Methods: To investigate whether this reduced binding effect of E297A/E303A/D382A is specific for bTSH we determined the triple mutants cAMP production after stimulation with human TSH (hTSH) and the hTSH analog TR1401 that differs from hTSH by four additional positively charged amino acids, which also exist in bTSH.
Results: Indeed, comparable to bTSH the cAMP signal of the triple mutant was reduced after stimulation with TR1401, in contrast to treatment with hTSH. Moreover, we investigated the hTSH TR1401 binding of several amino acid substitutions at the three hinge positions in comparison to bTSH binding. Most of the mutants showed a TR1401 binding comparable to bTSH binding.
Conclusions: Therefore, our data indicate that the negative charge of positions E297 and D382 is not only essential for bTSH but also for hTSH analog TR1401 binding. Together with no role of these receptor residues in the interaction with wt hTSH, these findings suggest that positively charged amino acids of TR1401, also present in bTSH, are most likely involved in direct electrostatic interactions with negatively charged residues of the TSHR hinge region. This finding implies that the hinge region is a mediator of the superagonistic activity of bTSH and of the hTSH analog TR1401.