Abstract
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.
Highlights
Top of the second transmembrane helix, to Ala resulted in a 225-fold loss of potency for gonadotropin-releasing hormone (GnRH)
Taken Site-directed mutagenesis provides a means for identifying with previous data, they suggest a region of the GnRH receptor residues involved in ligand binding
In COS-1 cells transiently expressing the [Ala102]GnRH receptors (GnRHRs), the EC50 for GnRH-stimulated inositol phosphate production was increased by a mean of 225-fold, compared with the wildtype receptor (Table I)
Summary
GnRH, gonadotropin-releasing hormone; GnRHR, GnRH receptor; GnRH-OH, GnRH free acid. we show that the enhancement of potency conferred by Asn102 is selective for peptides with a glycinamide moiety at the C terminus, while the corresponding ethylamide derivatives are less dependent on Asn102, indicating an important role for this residue in the docking of the C-terminal glycinamide of the ligand.
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