Molecular basis of hypogonadotropic hypogonadism:: Restoration of mutant (E90K) GnRH receptor function by a deletion at a distant site

GnRH regulates the synthesis and release of pituitary gonadotropins. Mutations in the human GnRH receptor (hGnRHR) gene have been reported in families with hypogonadotropic hypogonadism. Our group recently described a novel homozygous (EK)-K-90 mutation of the hGnRHR in two siblings with the complete form of hypogonadotropic hypogonadism. In the present study, mutational analysis of the (EK)-K-90 substitution was performed to assess the functional role of this particular residue, which is located in the second transmembrane helix of the hGnRHR. Although E-90 is highly conserved in all other known mammalian GnRH receptors, this residue has not been previously implicated in GnRH binding and/or GnRHR activation. Transient expression of the mutant (EK)-K-90 receptor in COS-7 cells resulted in a virtual abolition of GnRH agonist binding and agonist-stimulated phosphoinositide turnover, initially suggesting that E90 may be essential for GnRH binding. Furthermore, incubation with 1 muM of different GnRH agonists (D-Trp(6)-GnRH, GnRH, leuprolide, Catfish-1 GnRH, Catfish-2 GnRH, D-Lys(6)-Pro(9)-EA-GnRH, DesGly(10)-GnRH, D-Trp(6)Pro(9)-EA-GnRH, Buserelin, and D-Lys(6)-GnRH) or antagonists (Antide and "Nal-Arg") did not result in elevated inositol phosphate production from cells expressing the (EK)-K-90 mutant. To examine the role of a site known to suppress hGnRHR function, mutants with deletion of K-191 (DeltaK(191)) from the hGnRHR and/or addition of catfish GnRHR intracellular carboxyl-terminal tail (cfCtail) to hGnRHR were prepared. Exposure to the GnRH analog Buserelin resulted in a significant increase in total inositol phosphate production in cells expressing the hGnRHR-cfCtail, hGnRHR(DeltaK(191)) and hGnRHR(DeltaK(191))-cfCtail. Activation of intracellular signaling in response to Buserelin was restored by deletion of K-191 from the (EK)-K-90 mutant receptor but minimally by addition of the catfish GnRHR carboxyl-terminal tail. There were no significant differences in total inositol phosphate production between the chimeric receptors bearing the DeltaK(191) or the (EK)-K-90/DeltaK(191) modifications. All but the ((EK)-K-90) and ((EK)-K-90)-efCtail altered receptors were membrane expressed as disclosed by Western blot analysis of epitope-tagged receptors. This study provides evidence that the (EK)-K-90 mutation impairs hGnRHR-effector coupling. The observation that sequence modifications that enhance surface expression of the receptor restore function, presents the possibility that loss of surface expression may underlie the severe phenotype exhibited by hypogonadotropic hypogonadism patients bearing this mutational defect.