Received for publication August 10, 2007.
Revised September 19, 2007.
Accepted for publication October 16, 2007.

Identification of a Novel Ligand Binding Residue Arg^38(1.35) in the Human GnRH Receptor

Abstract

Delineation of peptide ligand binding sites is of fundamental importance in rational drug design and in understanding ligand-induced receptor activation. Molecular modeling and ligand docking to previously experimentally identified binding sites revealed a putative novel interaction between the C-terminus of gonadotropin-releasing hormone (GnRH) and Arg^38(1.35), located at the extracellular end of transmembrane domain (TM) 1 of the human GnRH receptor. Mutation of Arg^38(1.35) to Ala resulted in 989- and 1268-fold reduction in affinity for GnRH I and GnRH II, the two endogenous ligands. Conservative mutation of Arg^38(1.35) to Lys had less effect, giving reduced affinities of GnRH I and GnRH II by 24- and 54-fold. To test whether Arg^38(1.35) interacts with the C-terminal Gly¹⁰-NH₂ of GnRH, binding of GnRH analogs with substitution of the C-terminal glycinamide with ethylamide ([Pro⁹-NHEt]GnRH) was studied with wild-type and Arg^38(1.35) mutant receptors. Mutation of Arg^38(1.35) to Lys or Ala had much smaller effect on receptor affinity for [Pro⁹-NHEt]GnRH analogs and no effect on binding affinity of peptide antagonist cetrorelix. In parallel with the decreased affinity, the mutants also gave a decreased potency to GnRH-elicited inositol phosphate (IP) responses. The mutants had similar effects on [Pro⁹-NHEt]GnRH-elicited IP responses as that of the parent GnRHs. These findings indicate that Arg^38(1.35) of the GnRH receptor is essential for high-affinity binding of GnRH agonists and stabilizing the receptor active conformation. The mutagenesis results support the prediction of molecular modeling that Arg^38(1.35) interacts with the C-terminal glycinamide and likely forms hydrogen-bonds with the backbone carbonyl of Pro⁹ and Gly¹⁰-NH₂ of GnRH.

Key words: Gonadotropins, Gq/11 family, Structure-activity relationships and modeling, Func. analysis receptor/ion channel mutants

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