Functional Importance of Transmembrane Helix 6 Trp and Exoloop 3 Val of Rat Gonadotropin-Releasing Hormone Receptor

Previous studies have established that the interaction of gonadotropin-releasing hormone (GnRH) with its receptor (GnRHR) would require partial entry of the Nand C-terminal regions of ligand into the transmembrane core. The functional significance of the conserved aromatic residue Trp present in the transmembrane helix 6, and Val located in exoloop 3 of the rat GnRHR was investigated by mutagenesis followed by expression in Chinese hamster ovary-K1 cells. Compared with wildtype, substitution of Trp with Ser or Arg resulted in a marked reduction or total abolition, respectively, of ligand binding and, in both cases, abrogation of GnRH-induced inositol phosphate production. A total absence of functionality was observed when Val was simply replaced with Ala. Mention should be made that an expression of all mutated and wild-type receptor proteins was observed. Interestingly, the double mutant [TrpArg/ValAla]GnRHR restored Bmax to wild type (504 6 43 versus 541 6 41 fmol/mg protein), but with a diminished affinity (4.95 6 1.05 versus 0.94 6 0.35 nM), and GnRH failed to induce inositol phosphate. No influence of the mutations was seen on internalization of the receptor. The three-dimensional model of GnRH binding to the rat GnRHR was built predicting that Trp is buried at 20 Å in the transmembrane core of the receptor, directly in contact with Trp of GnRH. In contrast, Val is located in a region that cannot be precisely defined at the extracellular end of transmembrane helix 7. Although models cannot provide any clue concerning the observed interactivity between the two distal residues, altogether these data reveal the functional importance of both GnRHR Trp and Val and suggest that Trp, interacting with GnRH Trp, represents the bottom of the binding pocket. The effects of gonadotropin-releasing hormone (GnRH), a decapeptidic neurohormone secreted by the hypothalamus, are mediated by a specific receptor present on the cell surface of anterior pituitary gonadotropes (Counis, 1999). The cloning of cDNAs from various species and the amino acid sequence analysis have confirmed that the GnRH receptor (GnRHR) possesses the typical structural features of members of the G-protein-coupled receptor (GPCR) superfamily, which include seven membrane-spanning helices connected with three intracellular and three extracellular loops (Stojilkovic et al., 1994). However, the GnRHR exhibits unique structural features, such as the replacement of the conserved DRY sequence in the second intracellular loop by DRS, the reciprocal exchange of the highly conserved Asp in transmembrane helix (TMH) 2 and Asn in TMH7, and, most striking, the lack of a cytoplasmic C-terminal tail, which accounts for the slow internalization postactivation and, together with a relatively short N-terminal sequence, its reduced size (327 amino acids in rat and mouse; 328 in human, porcine, bovine, and ovine). Due to the central role of GnRH in reproductive physiology and the large range of clinical applications for both agonist and antagonist analogs (Conn and Crowley, 1994), an indepth knowledge of the GnRHR characteristics and ligand interaction is required. Site-directed mutagenesis has allowed the identification of several amino acid residues of the GnRHR that are functionally important (for binding and/or transduction, or internalization). In particular, evidence has been obtained that the binding site of GnRHR, like other GPCRs for peptide ligands, involves the extracellular domains as well as the transmembrane domains of the receptor. Specifically, His of GnRH has the potential to interact with This work was supported by grants from the Centre National de la Recherche Scientifique, the Université Pierre et Marie Curie and the Association pour la Recherche sur le Cancer (contract 1329). S.C. is recipient of a grant from the Ministère de l’Education Nationale, de la Recherche et de la Technologie. ABBREVIATIONS: GnRH, gonadotropin-releasing hormone (pyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2); GnRHR, GnRH receptor; TMH, transmembrane helix; GnRH-Ag, [His-DTyr]GnRH; GPCR, G protein-coupled receptor; LH, luteinizing hormone; FSH, follicle-stimulating hormone; TSH, thyrotropin-stimulating hormone; EGFP, enhanced green fluorescent protein; CHO, Chinese hamster ovary; PCR, polymerase chain reaction; ConA, concanavalin A; IP, inositol phosphate. 0026-895X/00/030625-09$3.00/0 Copyright © The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 57:625–633 (2000). 625 at A PE T Jornals on Jne 6, 2017 m oharm .aspeurnals.org D ow nladed from Lys present near the top of TMH3 (Zhou et al., 1995), Arg of GnRH with Glu (mouse, rat), or Asp (human, porcine, ovine, bovine) of exoloop 3 (Flanagan et al., 1994) and the C-terminal glycinamide with Asn located at the apex of TMH2 (Davidson et al., 1996). Altogether, these data are consistent with earlier studies related to the GnRH structure-activity demonstrating that the Nand C-terminal region are the most important for binding and receptor activation, whereas the central region (residues 5, 6, and 7) are most involved in conferring flexibility to the molecule (Karten and Rivier, 1986) and indicate that GnRH would interact with its receptor in a hairpin structure, with both the N and C tails partially penetrating the TMH bundle of its receptor. The transmembrane core of the GnRHR, like other GPCRs, is highly hydrophobic and contains conserved aromatic residues on helices 4, 5, 6, and 7, some of which have been shown to be critical determinants for ligand-receptor interaction in a large range of GPCRs (Trumpp-Kallmeyer et al., 1992). An implication of aromatic amino acids, and particularly Trp and Tyr, in the recognition site of GnRHR has been suggested earlier by Keinan and Hazum (1985) using a specific chemical approach. Consistently, aromatic residues Tyr (human), present in TMH6, and Tyr (mouse), present in TMH7, have been recently shown to be functionally essential in GnRH binding and/or signal transduction and internalization (Arora et al., 1996; Layman et al., 1998). The comparison of the primary sequence of a large number of GPCRs (Probst et al., 1992), including all known mammalian and nonmammalian GnRHR, has emphasized the highly conserved position of the Trp in both TMH4 and TMH6. Nevertheless, whereas the TMH4 Trp displayed total conservation among GPCRs, TMH6 Trp appeared essentially absent in odorant receptors and, most interestingly, in all members of the glycoprotein hormones receptor family (see Fig. 1), the ligand of which consists of large, complex hormones [luteinizing hormone (LH), follicle-stimulating hormone (FSH), or thyrotropin-stimulating hormone (TSH)], which do not seem to enter the TMH core. Thus, we hypothesized that the TMH6 Trp residue could be of specific importance in GnRHR func-