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Two Mutations in Extracellular Loop 2 of the Human GnRH Receptor Convert an Antagonist to an Agonist

Medical Research Council Human Reproductive Sciences Unit (T.R.O., R.P.M.), Edinburgh EH3 9ET, United Kingdom; Departments of Clinical Laboratory Sciences (T.R.O., B.E.T., C.A.F., R.P.M.) and Medicine (C.A.F.), University of Cape Town Medical School, Observatory 7925, South Africa; Indiana University School of Medicine (R.W.R.), Indianapolis, Indiana 46202; and Department of Molecular and Cellular Biology (N.I.), University of Cape Town, Rondebosch 7700, South Africa

Address all correspondence and requests for reprints to: Robert P. Millar, Medical Research Council Human Reproductive Sciences Unit, 37 Chalmers Street, Edinburgh EH3 9ET, United Kingdom. E-mail: r.millar{at}hrsu.mrc.ac.uk.

GnRH regulates the reproductive system through cognate G protein-coupled receptors in vertebrates. Certain GnRH analogs that are antagonists at mammalian receptors behave as agonists at Xenopus laevis and chicken receptors. This phenomenon provides the opportunity to elucidate interactions and the mechanism underlying receptor activation. A D-Lys(iPr) in position 6 of the mammalian GnRH receptor antagonist is required for this agonist activity (inositol phosphate production) in the chicken and X. laevis GnRH receptors. Chimeric receptors, in which extracellular loop domains of the human GnRH receptor were substituted with the equivalent domains of the X. laevis GnRH receptor, identified extracellular loop 2 as the determinant for agonist activity of one of the mammalian antagonists: antagonist 135-18. Site-directed mutagenesis of nine nonconserved residues in the C-terminal domain of extracellular loop 2 of the human GnRH receptor showed that a minimum of two mutations (Val^5.24(197)Ala and Trp^5.32(205)His) is needed in this region for agonist activity of antagonist 135-18. Agonist activity of antagonist 135-18 was markedly decreased by low pH (<7.0) compared with GnRH agonists. These findings indicate that D-Lys(iPr)⁶ forms a charge-supported hydrogen bond with His^5.32(205) to stabilize the receptor in the active conformation. This discovery highlights the importance of EL-2 in ligand binding and receptor activation in G protein-coupled receptors.

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