Conserved amino acid residues that are important for ligand binding in the type I gonadotropin-releasing hormone (GnRH) receptor are required for high potency of GnRH II at the type II GnRH receptor

GnRH I regulates reproduction. A second form, designated GnRH II, selectively binds type II GnRH receptors. Amino acids of the type I GnRH receptor required for binding of GnRH I (Asp^2.61(98), Asn ^2.65(102), and Lys^3.32(121)) are conserved in the type II GnRH receptor, but their roles in receptor function are unknown. We have delineated their functions using mutagenesis, signaling and binding assays, immunoblotting, and computational modeling. Mutating Asp^2.61(97) to Glu or Ala, Asn^2.65(101) to Ala, or Lys^3.32(120) to Gln decreased potency of GnRH II-stimulated inositol phosphate production. Consistent with proposed roles in ligand recognition, mutations eliminated measurable binding of GnRH II, whereas expression of mutant receptors was not decreased. In detailed analysis of how these residues affect ligand-dependent signaling, [Trp²]-GnRH I showed lesser decreases in potency than GnRH I at the Asp^2.61(97)Glu mutant. In contrast, [Trp²]-GnRH II showed the same loss of potency as GnRH II at this mutant. This suggests that Asp^2.61(97) contributes to recognition of His² of GnRH I, but not of GnRH II. GnRH II showed a large decrease in potency at the Asn ^2.65(101)Ala mutant compared with analogs lacking the C=O group of Gly¹⁰NH₂. This suggests that Asn^2.65(101) recognizes Gly¹⁰NH₂ of GnRH II. GnRH agonists showed large decreases in potency at the Lys^3.32(120)Gln mutant, but antagonist activity was unaffected. This suggests that Lys^3.32(120) recognizes agonists, but not antagonists, as in the type I receptor. These data indicate that roles of conserved residues are similar, but not identical, in the type I and II GnRH receptors.