Human placental gonadotrophin-releasing hormone (GnRH) binding sites: I. Characterization, properties and ligand specificity

Abstract

Radioiodinated gonadotrophin-releasing hormone tracers were prepared from mammalian (m GnRH), salmon (s GnRH), lamprey (l GnRH) and the two forms of chicken GnRH (ch GnRH I and ch GnRH II), and also from the GnRH agonist (GnRH A) analogues, Buserelin ([D-Ser(tBu) 6] 1–9 GnRH ethylamide) and Tryptorelin ([D-Trp 6 GnRH]1–9 ethylamide) and a GnRH antagonist (GnRH, ANT [Ac 3, 4-dehydro-Pro 1, D-p-F-Phe 2, D-Trp 3,6 LHRH). Specific binding of hormone tracers was compared in homogenates and membrane fractions from human placenta and rat pituitary. GnRH agonist tracers bound readily to pituitary and placental binding sites. Binding of m GnRH to rat pituitary membranes was low compared to agonist binding, whereas other GnRH iso forms were not bound. Binding of 125I-labelled m GnRH to human placental membranes was also low compared to that of Buserelin, and l GnRH and ch GnRHI tracers bound poorly. However, human placental membranes bounds GnRH and ch GnRH II to the same extent as GnRH A. Studies of the inactivation of GnRH tracers following incubation with rat pituitary and human placental membranes demonstrated that, although GnRH isoforms were degraded at different rates in these tissues, the differential ability of GnRH isoforms to bind to pituitary or placental binding sites was not related to differences in degradation of tracers, but rather to differences in ligand specificity. Specific binding of 125I-labelled GnRH agonists (GnRH A) and mammalian GnRH(m GnRH), s GnRH and ch GnRH II tracers to human placental membrane fractions increased linearly with increasing membrane protein at low concentrations. Binding was dependent on both the duration and temperature of incubation, and pH profiles of 125-labelled GnRHA, s GnRh and ch GnRH II binding to placental membranes were similar. Once bound s GnRH formed a tighter complex with placental receptors than GnRH A, though 125I-labelled s GnRH was inactivated more rapidly than agonist tracer during incubation with placental membranes. Binding of GnRH tracers was specific for molecules with the GnRH structure. Deletions of amino acid residues at positions 1–3 and/or deamidation at Gly 10 reduced binding potencies for both human placental and rat pituitary binding sites, indicating that both ends of the GnRH molecule were required for optimal binding. Modifications which conferred increased agonist activity led to markedly increased receptor binding potency in the rat pituitary, but only slightly increased potency for placental receptors. In contrast, GnRH antagonist analogues had increased potency towards pituitary receptors, but much reduced potency towards human placental binding sites. These studies highlight further the differences between human placental and rat pituitary GnRH receptors.