Dependence of agonist activation on an aromatic moiety in the DPLIY motif of the gonadotropin-releasing hormone receptor

In the GnRH receptor, the NPX(2-3)Y motif that is present in the seventh transmembrane helix of most G protein-coupled receptors is unusual in containing Asp instead of Asn but retains the highly conserved Tyr residue, The importance of this aromatic residue in the DPLIY sequence of the GnRH receptor function was analyzed by replacing Tyr(322) with Ala or Phe residues, The Y(322)A mutant receptor expressed in COS-7 cells had high agonist binding affinity, but its ability to interact with G protein(s) and to activate inositol phosphate production in response to GnRH was abolished. Although functionally inactive, the Y(322)A, mutant receptor was internalized at about 50% of the rate of the wild type receptor in agonist-treated cells, When Tyr(322) was replaced with Phe to preserve its aromatic nature, the (YF)-F-322 mutant receptor displayed normal G protein activation and inositol phosphate responses to GnRH and was internalized in the same manner as the wild type receptor, These findings demonstrate that the aromatic moiety of the Tyr(322) component of the DPLIY motif in the GnRH receptor is a critical determinant of agonist-induced receptor activation and signal transduction.