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Identification of a Novel Ligand Binding Residue Arg^38(1.35) in the Human Gonadotropin-Releasing Hormone Receptor

MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, the Queen's Medical Research Institute, Edinburgh, United Kingdom (A.J.S., R.S., D.J.W., R.P.M., Z.L.L.); and Research Group for Receptor Biology, Institute of Infectious Disease and Molecular Medicine, Division of Medical Biochemistry, University of Cape Town Faculty of Health Sciences, Observatory 7925, Cape Town, South Africa (R.P.M.)

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 1 of the human GnRH receptor. Mutation of Arg^38(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 Arg^38(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 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 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 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 probably forms hydrogen bonds with the backbone carbonyl of Pro⁹ and Gly¹⁰-NH₂.

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				R. Lopez de Maturana, A. J. Pawson, Z.-L. Lu, L. Davidson, S. Maudsley, K. Morgan, S. P. Langdon, and R. P. Millar Gonadotropin-Releasing Hormone Analog Structural Determinants of Selectivity for Inhibition of Cell Growth: Support for the Concept of Ligand-Induced Selective Signaling Mol. Endocrinol., July 1, 2008; 22(7): 1711 - 1722. [Abstract] [Full Text] [PDF]