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Abstract

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 pY²⁸³, Y²⁸⁴, W²⁸⁹, Y²⁹⁰, W²⁹¹, and F²⁰² were exchanged to alanine and analyzed comprehensively in functional reporter gene and ligand binding assays. Whereas receptor mutants Y²⁸³A, Y²⁸¹A, and W²⁹¹A were capable of neither ligand binding nor signal transduction, mutants W²¹⁹A, Y²⁹⁰A, and F²⁹²A were functional: the F²⁹²A mutant behaved like wild-type receptor, while mutants W²⁸⁹A and Y²⁹⁰A 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-Trp⁶-GnRH (Triptorelin) binding, representing a superagonistic ligand, is in full accordance to available data. Furthermore, new interactions are proposed: pGlu¹ interacts with N²¹² in transmembrane helix 5, Tyr⁵ with Y²⁹⁰, and D-Trp⁶ with W²⁸⁹. The binding behavior of mutants W²⁸⁹A and Y²⁹⁰A corresponds to the proposed binding model for the antagonist Cetrorelix. In summary, our data as presented indicate that Y²⁹⁰ plays a key function in agonist but not antagonist binding.