Photoaffinity labeling of pituitary gonadotropin-releasing hormone receptors in goldfish (Carassius auratus)

Abstract

Receptors for GnRH were labeled by use of an iodinated (125I) photoreactive GnRH derivative [D-Lys6-azidobenzoyl]-GnRH. This derivative was found to bind to two classes of GnRH binding sites: high-affinity/low-capacity sites and low-affinity/high-capacity sites. The binding affinity of [D-Lys6-azidobenzoyl]-GnRH was found to be greater than that of D-Lys6-GnRH, but lower than a superactive fish GnRH agonist [D-Arg6, Trp7, Leu8, Pro9-NEt]-GnRH (sGnRH-A). Analysis of the photoaffinity-labeled goldfish pituitary GnRH receptors by SDS-PAGE and autoradiography indicated the presence of three labeled proteins displaceable by unlabeled sGnRH-A. The first and the most prominently labeled band was a 71 000-M(r) protein, the second a 51 000-M(r) protein, and the third a minor band of 130 000 M(r). Displacement characteristics of the 71 000- and 130 000-M(r) bands were consistent with those of the low-affinity binding sites; displacement of the iodinated ligand from these proteins was achieved only in the presence of 10-6 M sGnRH-A. The 51 000-M(r) band had characteristics similar to those of the high-affinity site; displacement of the labeled ligand was achieved in the presence of 10-9 M sGnRH-A. These findings provide for the first time some biochemical characterizations of pituitary GnRH receptors in a nonmammalian vertebrate.