Identification of Tyr^290(6.58) of the human gonadotropin- releasing hormone (GnRH) receptor as a contact residue for both GnRH I and GnRH II: Importance for high-affinity binding and receptor activation

Molecular modeling showed interactions of Tyr^290(6.58) in transmembrane domain 6 of the GnRH receptor with Tyr⁵ of GnRH I, and His⁵ of GnRH II. The wild-type receptor exhibited high affinity for [Phe⁵]GnRH I and [Tyr⁵]GnRH II, but 127- and 177-fold decreased affinity for [Ala⁵]GnRH I and [Ala⁵]GnRH II, indicating that the aromatic ring in position 5 is crucial for receptor binding. The receptor mutation Y290F decreased affinity for GnRH I, [Phe ⁵]GnRH I, GnRH II and [Tyr⁵]GnRH II, while Y290A and Y290L caused larger decreases, suggesting that both the para-OH and aromatic ring of Tyr^290(6.58) are important for binding of ligands with aromatic residues in position 5. Mutating Tyr^290(6.58) to Gln increased affinity for Tyr⁵-containing GnRH analogues 3-12-fold compared with the Y290A and Y290L mutants, suggesting a hydrogen-bond between Gln of the Y290Q mutant and Tyr⁵ of GnRH analogues. All mutations had small effects on affinity of GnRH analogues that lack an aromatic residue in position 5. These results support direct interactions of the Tyr^290(6.58) side chain with Tyr⁵ of GnRH I and His⁵ of GnRH II. Tyr ^290(6.58) mutations, except for Y290F, caused larger decreases in GnRH potency than affinity, indicating that an aromatic ring is important for the agonist-induced receptor conformational switch.