TY - JOUR T1 - Functional Importance of Transmembrane Helix 6 Trp<sup>279</sup>and Exoloop 3 Val<sup>299</sup> of Rat Gonadotropin-Releasing Hormone Receptor JF - Molecular Pharmacology JO - Mol Pharmacol SP - 625 LP - 633 DO - 10.1124/mol.57.3.625 VL - 57 IS - 3 AU - Stéphanie Chauvin AU - Annette Bérault AU - Yannick Lerrant AU - Marcel Hibert AU - Raymond Counis Y1 - 2000/03/01 UR - http://molpharm.aspetjournals.org/content/57/3/625.abstract N2 - Previous studies have established that the interaction of gonadotropin-releasing hormone (GnRH) with its receptor (GnRHR) would require partial entry of the N- and C-terminal regions of ligand into the transmembrane core. The functional significance of the conserved aromatic residue Trp279 present in the transmembrane helix 6, and Val299 located in exoloop 3 of the rat GnRHR was investigated by mutagenesis followed by expression in Chinese hamster ovary-K1 cells. Compared with wild-type, substitution of Trp279 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 Val299 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 [Trp279Arg/Val299Ala]GnRHR restoredB max to wild type (504 ± 43 versus 541 ± 41 fmol/mg protein), but with a diminished affinity (4.95 ± 1.05 versus 0.94 ± 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 Trp279is buried at 20 Å in the transmembrane core of the receptor, directly in contact with Trp3 of GnRH. In contrast, Val299 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 Trp279 and Val299 and suggest that Trp279, interacting with GnRH Trp3, represents the bottom of the binding pocket. ER -