PT  - JOURNAL ARTICLE
AU  - Alan J. Stewart
AU  - Robin Sellar
AU  - Donald J. Wilson
AU  - Robert P. Millar
AU  - Zhi-Liang Lu
TI  - Identification of a Novel Ligand Binding Residue Arg<sup>38(1.35)</sup> in the Human Gonadotropin-Releasing Hormone Receptor
AID  - 10.1124/mol.107.040816
DP  - 2008 Jan 01
TA  - Molecular Pharmacology
PG  - 75--81
VI  - 73
IP  - 1
4099  - http://molpharm.aspetjournals.org/content/73/1/75.short
4100  - http://molpharm.aspetjournals.org/content/73/1/75.full
SO  - Mol Pharmacol2008 Jan 01; 73
AB  - 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 Arg38(1.35), located at the extracellular end of transmembrane domain 1 of the human GnRH receptor. Mutation of Arg38(1.35) to alanine resulted in 989- and 1268-fold reduction in affinity for GnRH I and GnRH II, respectively, the two endogenous ligands. Conservative mutation of Arg38(1.35) to lysine had less effect, giving reduced affinities of GnRH I and GnRH II by 24- and 54-fold, respectively. To test whether Arg38(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 Arg38(1.35) mutant receptors. Mutation of Arg38(1.35) to lysine or alanine 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 mutant receptors had effects on [Pro9-NHEt]GnRH-elicited IP responses similar to that of the parent GnRHs. These findings indicate that Arg38(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 Arg38(1.35) interacts with the C-terminal glycinamide and probably forms hydrogen bonds with the backbone carbonyl of Pro9 and Gly10-NH2. The American Society for Pharmacology and Experimental Therapeutics