Asn102 of the gonadotropin-releasing hormone receptor is a critical determinant of potency for agonists containing C-terminal glycinamide.
We demonstrate a critical role for Asn102 of the human gonadotropin-releasing hormone (GnRH) receptor in the binding of GnRH. Mutation of Asn102, located at the top of the second transmembrane helix, to Ala resulted in a 225-fold loss of potency for GnRH. Eight GnRH analogs, all containing glycinamide C termini like GnRH, showed similar losses of potency between 95- and 750-fold for the [Ala102]GnRHR, compared with wild-type receptor. In contrast, four GnRH analogs that had ethylamide in place of the C-terminal glycinamide residue, showed much smaller decreases in potency between 2.4- and 11-fold. In comparisons of three agonist pairs, differing only at the C terminus, glycinamide derivatives showed an 11-20-fold greater loss of potency for the mutant receptor than their respective ethylamide derivatives. Thus Asn102 is a critical determinant of potency specifically for ligands with C-terminal glycinamide, while ligands with C-terminal ethylamide are less dependent on Asn102. These findings indicate a role for Asn102 in the docking of the glycinamide C terminus and are consistent with hydrogen bonding of the Asn102 side chain with the C-terminal amide moiety. Taken with previous data, they suggest a region of the GnRH receptor formed by the top of helices 2 and 7 as a binding pocket for the C-terminal part of the ligand.
Davidson
J S
JS
Department of Chemical Pathology, University of Cape Town Medical School, Observatory 7925, South Africa.
McArdle
C A
CA
Davies
P
P
Elario
R
R
Flanagan
C A
CA
Millar
R P
RP
D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry*; D001665:Binding Sites; D005998:Glycine / Q000031:analogs & derivatives / Q000737:chemistry; D007987:Gonadotropin-Releasing Hormone / Q000737:chemistry*; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011966:Receptors, LHRH / Q000737:chemistry*; D015398:Signal Transduction; D013329:Structure-Activity Relationship
D007295:Inositol Phosphates; D008024:Ligands; D009838:Oligodeoxyribonucleotides; D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone; C018556:glycine amide; D001216:Asparagine; D005998:Glycine