Involvement of Amino Acids Flanking Glu7.32 of the Gonadotropin-releasing Hormone Receptor in the Selectivity of Antagonists

  • Wang, Chengbing (Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University) ;
  • Oh, Da Young (Graduate School of Medicine, Korea University) ;
  • Maiti, Kaushik (Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University) ;
  • Kwon, Hyuk Bang (Hormone Research Center, School of Biological Sciences and Technology, Chonnam National University) ;
  • Cheon, Jun (Department of Urological Oncology, Korea University Hospital) ;
  • Hwang, Jong-Ik (Graduate School of Medicine, Korea University) ;
  • Seong, Jae Young (Graduate School of Medicine, Korea University)
  • Received : 2007.07.03
  • Accepted : 2007.09.11
  • Published : 2008.02.29


The Glu/$Asp^{7.32}$ residue in extracellular loop 3 of the mammalian type-I gonadotropin-releasing hormone receptor (GnRHR) interacts with $Arg^8$ of GnRH-I, conferring preferential ligand selectivity for GnRH-I over GnRH-II. Previously, we demonstrated that the residues (Ser and Pro) flanking Glu/$Asp^{7.32}$ also play a role in the differential agonist selectivity of mammalian and non-mammalian GnRHRs. In this study, we examined the differential antagonist selectivity of wild type and mutant GnRHRs in which the Ser and Pro residues were changed. Cetrorelix, a GnRH-I antagonist, and Trptorelix-2, a GnRH-II antagonist, exhibited high selectivity for mammalian type-I and non-mammalian GnRHRs, respectively. The inhibitory activities of the antagonists were dependent on agonist concentration and subtype. Rat GnRHR in which the Ser-Glu-Pro (SEP) motif was changed to Pro-Glu-Val (PEV) or Pro-Glu-Ser (PES) had increased sensitivity to Trptorelix-2 but decreased sensitivity to Cetrorelix. Mutant bullfrog GnRHR-1 with the SEP motif had the reverse antagonist selectivity, with reduced sensitivity to Trptorelix-2 but increased sensitivity to Cetrorelix. These findings indicate that the residues flanking $Glu^{7.32}$ are important for antagonist as well as agonist selectivity.


Anatgonist Selectivity;Extracellular Loop 3(ECL3);G Protein-coupled Receptor (GPCR);Gonadotropin-releasing Hormone (GnRh);GnRH Receptor;Ser-Glu-Pro (SEP) Motif


Supported by : Ministry of Health & Welfare, Korea Research Foundation


  1. Conn, P.M. and Crowley, W.F., Jr. (1994). Gonadotropinreleasing hormone and its analogs. Annu. Rev. Med. 45, 391-405
  2. Hoffmann, S.H., ter Laak, T., Kuhne, R., Reilander, H., and Beckers, T. (2000). Residues within transmembrane helices 2 and 5 of the human gonadotropin-releasing hormone receptor contribute to agonist and antagonist binding. Mol. Endocrinol. 14, 1099-1115
  3. Horvat, R.D., Roess, D.A., Nelson, S.E., Barisas, B.G., and Clay, C.M. (2001). Binding of agonist but not antagonist leads to fluorescence resonance energy transfer between intrinsically fluorescent gonadotropin-releasing hormone receptors. Mol. Endocrinol. 15, 695-703
  4. Illing, N., Troskie, B.E., Nahorniak, C.S., Hapgood, J.P., Peter, R.E., and Millar, R.P. (1999). Two gonadotropin-releasing hormone receptor subtypes with distinct ligand selectivity and differential distribution in brain and pituitary in the goldfish (Carassius auratus). Proc. Natl. Acad. Sci. USA 96, 2526-2531
  5. Millar, R., Lowe, S., Conklin, D., Pawson, A., Maudsley, S., Troskie, B., Ott, T., Millar, M., Lincoln, G., Sellar, R., et al. (2001). A novel mammalian receptor for the evolutionarily conserved type II GnRH. Proc. Natl. Acad. Sci. USA 98, 9636-9641
  6. Sealfon, S.C., Weinstein, H., and Millar, R.P. (1997). Molecular mechanisms of ligand interaction with the gonadotropinreleasing hormone receptor. Endocr. Rev. 18, 180-205
  7. Seong, J.Y., Wang, L., Oh da, Y., Yun, O., Maiti, K., Li, J.H., Soh, J.M., Choi, H.S., Kim, K., Vaudry, H., et al. (2003). Ala/Thr(201) in extracellular loop 2 and Leu/Phe (290) in transmembrane domain 6 of type 1 frog gonadotropin-releasing hormone receptor confer differential ligand sensitivity and signal transduction. Endocrinology 144, 454-466
  8. Matsuo, H., Baba, Y., Nair, R.M., Arimura, A., and Schally, A. V. (1971). Structure of the porcine LH- and FSH-releasing hormone. I. The proposed amino acid sequence. Biochem. Biophys. Res. Commun. 43, 1334-1339
  9. Becker, O.M., Shacham, S., Marantz, Y., and Noiman, S. (2003). Modeling the 3D structure of GPCRs: advances and application to drug discovery. Curr. Opin. Drug. Discov. Devel. 6, 353-361
  10. Hovelmann, S., Hoffmann, S.H., Kuhne, R., ter Laak, T., Reilander, H., and Beckers, T. (2002). Impact of aromatic residues within transmembrane helix 6 of the human gonadotropin- releasing hormone receptor upon agonist and antagonist binding. Biochemistry 41, 1129-1136
  11. Maiti, K., Li, J.H., Wang, A.F., Acharjee, S., Kim, W.P., Im, W.B., Kwon, H.B., and Seong, J. Y. (2003). GnRH-II analogs for selective activation and inhibition of non-mammalian and type-II mammalian GnRH receptors. Mol. Cells 16, 173-179
  12. Stojilkovic, S.S., Reinhart, J., and Catt, K.J. (1994). Gonadotropin-releasing hormone receptors: structure and signal transduction pathways. Endocr. Rev. 15, 462-499
  13. Horvath, J.E., Bajo, A.M., Schally, A.V., Kovacs, M., Herbert, F., and Groot, K. (2002). Effects of long-term treatment with the luteinizing hormone-releasing hormone (LHRH) agonist Decapeptyl and the LHRH antagonist Cetrorelix on the levels of pituitary LHRH receptors and their mRNA expression in rats. Proc. Natl. Acad. Sci. USA 99, 15048-15053
  14. Karten, M.J. and Rivier, J.E. (1986). Gonadotropin-releasing hormone analog design. Structure-function studies toward the development of agonists and antagonists: rationale and perspective. Endocr. Rev. 7, 44-66
  15. Wang, L., Bogerd, J., Choi, H.S., Seong, J.Y., Soh, J.M., Chun, S.Y., Blomenrohr, M., Troskie, B.E., Millar, R.P., Yu, W.H., et al. (2001). Three distinct types of GnRH receptor characterized in the bullfrog. Proc. Natl. Acad. Sci. USA 98, 361-366
  16. Bissantz, C., Bernard, P., Hibert, M., and Rognan, D. (2003). Protein-based virtual screening of chemical databases. II. Are homology models of G-Protein Coupled Receptors suitable targets? Proteins 50, 5-25
  17. Millar, R.P., Lu, Z.L., Pawson, A.J., Flanagan, C.A., Morgan, K., and Maudsley, S.R. (2004). Gonadotropin-releasing hormone receptors. Endocr. Rev. 25, 235-275
  18. Pawson, A.J., Morgan, K., Maudsley, S.R., and Millar, R.P. (2003). Type II gonadotrophin-releasing hormone (GnRH-II) in reproductive biology. Reproduction 126, 271-278
  19. Powell, J.F., Zohar, Y., Elizur, A., Park, M., Fischer, W.H., Craig, A.G., Rivier, J.E., Lovejoy, D.A., and Sherwood, N.M. (1994). Three forms of gonadotropin-releasing hormone characterized from brains of one species. Proc. Natl. Acad. Sci. USA 91, 12081-12085
  20. Li, J. H., Choe, H., Wang, A.F., Maiti, K., Wang, C., Salam, A., Chun, S.Y., Lee, W.K., Kim, K., Kwon, H.B., et al. (2005). Extracellular loop 3 (EL3) and EL3-proximal transmembrane helix 7 of the mammalian type I and type II gonadotropin-releasing hormone (GnRH) receptors determine differential ligand selectivity to GnRH-I and GnRH-II. Mol. Pharmacol. 67, 1099-1110
  21. Blomenrohr, M., ter Laak, T., Kuhne, R., Beyermann, M., Hund, E., Bogerd, J., and Leurs, R. (2002). Chimaeric gonadotropin-releasing hormone (GnRH) peptides with improved affinity for the catfish (Clarias gariepinus) GnRH receptor. Biochem. J. 361, 515-523
  22. Fromme, B.J., Katz, A.A., Millar, R.P., and Flanagan, C.A. (2004). Pro7.33(303) of the human GnRH receptor regulates selective binding of mammalian GnRH. Mol. Cell. Endocrinol. 219, 47-59
  23. Wang, A.F., Li, J.H., Maiti, K., Kim, W.P., Kang, H.M., Seong, J.Y., and Kwon, H.B. (2003). Preferential ligand selectivity of the monkey type-II gonadotropin-releasing hormone (GnRH) receptor for GnRH-2 and its analogs. Mol. Cell. Endocrinol. 209, 33-42
  24. Seeburg, P.H., Mason, A.J., Stewart, T.A., and Nikolics, K. (1987). The mammalian GnRH gene and its pivotal role in reproduction. Recent. Prog. Horm. Res. 43, 69-98
  25. Wang, C., Yun, O., Maiti, K., Oh D,Y., Kim, K.K., Chae, C.H., Lee, C.J., Seong, J.Y., and Kwon, H.B. (2004). Position of Pro and Ser near Glu7.32 in the extracellular loop 3 of mammalian and non-mammalian gonadotropin-releasing hormone (GnRH) receptors is a critical determinant for differential ligand selectivity for mammalian GnRH and chicken GnRH-II. Mol. Endocrinol. 18, 105-116
  26. White, R.B., Eisen, J.A., Kasten, T.L., and Fernald, R.D. (1998). Second gene for gonadotropin-releasing hormone in humans. Proc. Natl. Acad. Sci. USA 95, 305-309
  27. Fernald, R.D. and White, R.B. (1999). Gonadotropin-releasing hormone genes: phylogeny, structure, and functions. Front. Neuroendocrinol. 20, 224-240
  28. Nelson, S., Horvat, R.D., Malvey, J., Roess, D.A., Barisas, B.G., and Clay, C.M. (1999). Characterization of an intrinsically fluorescent gonadotropin-releasing hormone receptor and effects of ligand binding on receptor lateral diffusion. Endocrinology 140, 950-957
  29. Fromme, B.J., Katz, A.A., Roeske, R.W., Millar, R.P., and Flanagan, C.A. (2001). Role of aspartate7.32(302) of the human gonadotropin-releasing hormone receptor in stabilizing a high-affinity ligand conformation. Mol. Pharmacol. 60, 1280-1287
  30. Burgus, R., Butcher, M., Amoss, M., Ling, N., Monahan, M., Rivier, J., Fellows, R., Blackwell, R., Vale, W., and Guillemin, R. (1972). Primary structure of the ovine hypothalamic luteinizing hormone-releasing factor (LRF) (LH-hypothalamus-LRF-gas chromatography-mass spectrometrydecapeptide- Edman degradation). Proc. Natl. Acad. Sci. USA 69, 278-282
  31. Choi, H.J., Park, Y.G., and Kim, C.H. (2007). Lactosylceramide alpha2,3-sialyltransferase is induced via a PKC/ERK/CREB-dependent pathway in K562 human leukemia cells. Mol. Cells 23, 138-144
  32. Flanagan, C.A., Becker, II, Davidson, J.S., Wakefield, I.K., Zhou, W., Sealfon, S.C., and Millar, R.P. (1994). Glutamate 301 of the mouse gonadotropin-releasing hormone receptor confers specificity for arginine 8 of mammalian gonadotropin-releasing hormone. J. Biol. Chem. 269, 22636-22641
  33. Miyamoto, K., Hasegawa, Y., Nomura, M., Igarashi, M., Kangawa, K., and Matsuo, H. (1984). Identification of the second gonadotropin-releasing hormone in chicken hypothalamus: evidence that gonadotropin secretion is probably controlled by two distinct gonadotropin-releasing hormones in avian species. Proc. Natl. Acad. Sci. USA 81, 3874-3878
  34. Neill, J.D., Duck, L.W., Sellers, J.C., and Musgrove, L.C. (2001). A gonadotropin-releasing hormone (GnRH) receptor specific for GnRH II in primates. Biochem. Biophys. Res. Commun. 282, 1012-1018
  35. Oh, D.Y., Wang, L., Ahn, R.S., Park, J.Y., Seong, J.Y., and Kwon, H.B. (2003). Differential G protein coupling preference of mammalian and non-mammalian gonadotropin-releasing hormone receptors. Mol. Cell. Endocrinol. 205, 89-98
  36. Pfleger, K.D., Bogerd, J., and Millar, R.P. (2002). Conformational constraint of mammalian, chicken, and salmon GnRHs, but not GnRH II, enhances binding at mammalian and nonmammalian receptors: evidence for preconfiguration of GnRH II. Mol. Endocrinol. 16, 2155-2162
  37. Schally, A.V. (1999). LH-RH analogues: I. Their impact on reproductive medicine. Gynecol. Endocrinol. 13, 401-409
  38. Song, J.A., Oh, D.Y., Moon, J.S., Geum, D., Kwon, H.B., and Seong, J.Y. (2006). Involvement of the ser-glu-pro motif in ligand species-dependent desensitisation of the rat gonadotrophin-releasing hormone receptor. J. Neuroendocrinol. 18, 757-766