Impact of aromatic residues within transmembrane helix 6 of the human gonadotropin-releasing hormone receptor upon agonist and antagonist binding

To investigate the impact of aromatic residues within transmembrane helix 6 (TMH6) of the human gonadotropin-releasing hormone receptor (GnRH-R) on agonist and antagonist binding, residues Y-283, Y-284, W-289, Y-290, W-291, and F-202 were exchanged to alanine and analyzed comprehensively in functional reporter gene and ligand binding assays. Whereas receptor mutants Y(283)A, Y(281)A, and W(291)A were capable of neither ligand binding nor signal transduction, mutants W(219)A, Y(290)A, and F(292)A were functional: the F292A mutant behaved like wild-type receptor, while mutants W(289)A and Y(290)A differentiated between agonistic and antagonistic ligands. On the basis of the high-resolution X-ray structure of bovine rhodopsin as well as available data on GnRH-R mutants, models for ligand-receptor interactions are proposed. The model for D-Trp(6)-GnRH (Triptorelin) binding, representing a superagonistic ligand, is in full accordance to available data. Furthermore, new interactions are proposed: pGlu(1) interacts with N-212 in transmembrane helix 5, Tyr(5) with Y-290, and D-Trp(6) with W-289. The binding behavior of mutants W(289)A and Y(290)A corresponds to the proposed binding model for the antagonist Cetrorelix. In summary, our data as presented indicate that Y-290 plays a key function in agonist but not antagonist binding.