Endocrinology, Vol 135, 919-928, Copyright © 1994 by Endocrine Society

ARTICLES

Characterization of ovine growth hormone (oGH) and ovine placental lactogen (oPL) binding to fetal and adult hepatic tissue in sheep: evidence that oGH and oPL interact with a common receptor

BH Breier, B Funk, A Surus, GR Ambler, CA Wells, MJ Waters and PD Gluckman
Research Center for Developmental Medicine and Biology, University of Auckland School of Medicine, New Zealand.

The GH receptor (GHR) plays a key role in postnatal growth regulation. Although plasma concentrations of GH are high during fetal life, its role during fetal development is not well understood. Recent data suggest that GHR are present in fetal hepatic tissue as early as 51 days gestation. However, the levels of GHR expression are markedly lower in fetal hepatic tissue compared to postnatal values, and there are conflicting data suggesting that ovine placental lactogen (oPL) and oGH share a common receptor. Given the uncertainty about whether oPL acts via the oGHR or a distinct receptor, we performed ligand binding and affinity cross-linking studies on hepatic microsomal membranes from adult castrated male, pregnant female, and fetal sheep. Ligand binding assays at a constant concentration of membranes showed that [125I]oPL yielded consistently higher (P < 0.001) specific binding (59.5 +/- 6.4%, 30.5 +/- 5.7%, and 7.6 +/- 2.4% for castrated male, pregnant female, and fetal sheep, respectively) compared to [125I]oGH (17.8 +/- 4.7%, 5.0 +/- 1.6%, and 1.2 +/- 0.4% for castrated male, pregnant female, and fetal sheep, respectively). Cross-reactivity studies showed that unlabeled oPL was consistently more potent than unlabeled oGH in displacing either of the labeled ligands. The dissociation constant (Kd) for oPL binding ranged from 0.16-0.40 nM and was not changed by solubilization with Triton X-100. Equilibrium binding analysis for oGH showed lower affinity for hepatic microsomal membranes (Kd, 1.7-3.2 nM) in each of the three groups of animals. Affinity cross-linking of microsomal membranes from castrated male and pregnant female sheep liver showed four major cross-linked complexes with both [125I]oPL and [125I]oGH, with mol wt of 150, 97, 75, and 60 kilodaltons. All four bands were identified with both ligands. Unlabeled oPL showed markedly higher potency than unlabeled oGH in reducing the signal of the [125I]oPL cross-linked complexes, whereas unlabeled oGH and oPL showed comparable potencies in reducing the signal of the [125I]oGH complexes. Immunoprecipitation of detergent-solubilized hepatic microsomal membranes from pregnant and fetal sheep using a panel of monoclonal antibodies raised against the extracellular region of the rabbit GHR showed potent immunological recognition of the [125I]oPL-receptor complexes. We suggest that oGH and oPL bind to a common or a related receptor protein(s). It is possible that differences in receptor dimerization or association with other membrane proteins are the basis of the differences in affinity and biological actions of the two hormones.

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