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Vol. 57, Issue 3, 625-633, March 2000
Endocrinologie Cellulaire et Moléculaire de la Reproduction
(S.C., A.B., Y.L., R.C.), Université Pierre et Marie Curie,
Centre National de la Recherche Scientifique-ESA 7080, Paris,
France; and Laboratoire de Pharmacochimie de la Communication
Cellulaire (M.H.), Faculté de Pharmacie de Strasbourg, Illkirch,
France
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 restored
Bmax 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 Trp279
is 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.
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