Photoaffinity Labeling of Pituitary and Gonadal Receptors for Gonadotropin-Releasing Hormone

Abstract

A radioactive photoaffinity label for the GnRH receptor was prepared by derivatization of radiodinated [D-Lys6] des-Gly10-GnRH N-ethylamide with the heterobifunctional photolabile reagent N-hydroxysuccinimidyl-4-azido-benzoate. This high affinity photoreactive analog was employed for radiolabeling and characterization of pituitary GnRH receptors in rat, rabbit, mouse, sheep and cow adenohypophyses and gonadal GnRH receptors in the rat ovary and testis. In rat, rabbit and mouse pituitary glands, analysis of the GnRH receptor-ligand complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed 2 labeled components, both of which were displaced by unlabeled GnRH agonist and antagonist analogs. The larger receptor component was a relatively broad band, with MW in rat, rabbit and mouse of 59,000 .+-. 1900, 62,000 .+-. 700, and 60,000 .+-. 800, respectively. In the rat pituitary gland, the larger component was composed of 63,000 and 52,000 MW components, of which the latter was more heavily labeled and was predominant in purified pituitary gonadotrophs. The MW of the smaller components were 40,000 .+-. 1600, 43,000 .+-. 1200, and 41,000 .+-. 1000, respectively. In bovine and ovine pituitary glands, the photolabeled GnRH receptor was a single band with MW of 42,000 .+-. 1200 and 39,000 .+-. 500, respectively. In the rat ovary and testis, photolabeled GnRH receptors were similar to those in the rat pituitary gland, with 2 distinct components of 53,000 .+-. 1000 and 42,000 .+-. 1000 MW. Apparently, the pituitary receptors that mediate similar physiological actions of GnRH in different species possess broadly similar structural properties, with minor variations between species. The divergent actions of GnRH in different tissues of the same species, as in the rat pituitary and gonads, are expressed through receptors of similar structure.