Receptor-binding properties of gonadotropin-releasing hormone derivatives. Prolonged receptor occupancy and cell-surface localization of a potent antagonist analog.

The receptor-binding properties and in vitro biological effects of a highly active gonadotropin-releasing hormone (GnRH) antagonist, [N-acetyl-D-p-chloro-Phe1,2D-Trp3,D-Lys6,D-Ala10]GnRH, were compared with those of the GnRH superagonist analog, [D-Ala6] des-Gly10-GnRH-N-ethylamide. In rat pituitary particles and isolated pituitary cells, the 125I-labeled GnRH antagonist showed saturable high-affinity binding (Ka v 8.4 +/- 1.4 X 10(9) M-1) to the same receptor sites which bound the GnRH agonist. The rate of dissociation of the receptor-bound antagonist from pituitary particles and cells was extremely slow in comparison with that of the agonist ligand. Also, dissociation of the antagonist analog was incomplete, with a residual fraction of tightly bound ligand that was proportional to the duration of preincubation. The [D-Lys6]GnRH antagonist prevented GnRH-induced luteinizing hormone release during static incubation and superfusion of cultured pituitary cells, but in contrast to the agonist did not cause desensitization of the gonadotroph. Although the antagonist caused a prolonged reduction in available GnRH receptor sites, this was attributable to persistent occupancy by the slowly dissociating ligand rather than to receptor loss. Autoradiographic analysis of [D-Lys6]GnRH-antagonist uptake by cultured pituitary cells revealed that the peptide remained bound at the cell membrane for up to 2 h, in contrast with the rapid endocytosis of GnRH agonists. The slow dissociation of receptor-bound antagonist was consistent with its ability to cause sustained blockade of GnRH actions, and its prolonged cell-surface location suggests that receptor activation is necessary to initiate the rapid internalization of hormone-receptor complexes that is a feature of the agonist-stimulated gonadotroph.