Ruminant Placental Lactogens Act as Antagonists to Homologous Growth Hormone Receptors and as Agonists to Human or Rabbit Growth Hormone Receptors

Abstract

Growth hormone receptor (GHR)-mediated activity of ruminant placental lactogens (PLs) and ovine (o) GH was compared, using cells transfected with full size human (h), rabbit (rb), and oGHRs. All three PLs acted as agonists in heterologous bioassays, whereas in homologous bioassays in cells transfected with oGHRs they antagonized the oGH activity. Despite these differences, oGH and PLs bound with similar affinity to the oGHR extracellular domain (oGHR-ECD), indicating that the binding occurs through hormone site I. Gel filtration of complexes between oPL and oGHR-ECD showed a 1:1 stoichiometry, confirming this conclusion. The oPL T185D and bPL T188D, which exhibited weak biological activity mediated through GHRs, behaved as site I antagonists, whereas oPL G130R and bPL G133R formed a 1:1 complex with GHR-ECDs and bound to h/rb/oGHRECDs with affinity similar to that of wild-type oPL. They had no agonistic activity in all models transfected with h/rb and oGHRs, but were antagonistic to all of them. In conclusion, ruminant PLs antagonize the activity of oGH in homologous systems, because they cannot homodimerize oGHRs, whereas in heterologous systems they act as agonists. The structural analysis hints that minor differences in the sequence of the GHR-ECDs may account for this difference. Since the initial step in the activity transduced through cytokine/hemapoietic receptors family is receptor homodimerization or heterodimerization, we suggest that the question of homologous versus heterologous interactions should be reexamined.

Gh and igf-i increase leptin receptor expression in prepubertal pig ovaries: The role of leptin in steroid secretion and cell apoptosis

“…Ovine GH was prepared at the Institute of Biochemistry, Food Science and Nutrition (Rehovot, Israel) in the laboratory of Professor Gertler. Its biological activity was equal to that of human GH as documented using FDC-P1 cells stable transfected with rabbit GHR (Herman et al, 1999). IGF-I was prepared in the laboratory of Dr. Monsanto, and its biological activity was documented using undifferentiated mammary epithelial cells cultured in collagen, in serum-free medium (Peri et al, 1992) and mouse mammary cell line MME-L1 (Fine et al, 1997).…”

Mutations of ovine and bovine placental lactogens change, in different ways, the biological activity mediated through homologous and heterologous lactogenic receptors

“…We and others have also documented the fact that ruminant PLs are capable of activating either human (h) or rabbit (rb) growth hormone receptor (GHR)-mediated biological activities in several in vitro models (Byatt et al 1991. Recently, however, we have shown that oPL, bPL and cPL are inactive in homologous bioassays, namely in cells transfected with oGHRs, and even act as antagonists, in contrast to oGH, whose agonistic activity was equal to that of hGH (Herman et al 1999). Despite the differences in their biological activity, oGH and PLs bound with similar affinity to the oGHR extracellular domain (oGHR-ECD), indicating that the binding occurs through site 1 of the hormone.…”

“…The transfection and bioassay were carried out as described previously and the effects of the hormones and their analogues were determined by measuring the luciferase activity in cell lysates (Tchelet et al 1997, Herman et al 1999). …”

“…Two colonies were sequenced and confirmed to contain the mutation and no undesired misincorporation of nucleotides. Ovine PL-analogue T185F expression vector was constructed as described previously for oPL T185D (Herman et al 1999), using PCR technology. The sequences of the sense and antisense primers were 5 -GGAGATATACCATGGCACA GCATCCACC-3 and 5 -GCACTTAAGTATCCGG AGGTAGAAGTAAATTTTAC-3 respectively.…”

“…Bovine PL is glycosylated, though the glycosylation is not important for its biological activity (Byatt et al 1992). We and others have prepared recombinant oPL (Colosi et al 1989, Sakal et al 1997, bPL (Krivi et al 1989) and, recently, also cPL (Sakal et al 1998), as well as several bPL and oPL, mutants (Vashdi-Elberg et al 1995, Herman et al 1999. We and others have also documented the fact that ruminant PLs are capable of activating either human (h) or rabbit (rb) growth hormone receptor (GHR)-mediated biological activities in several in vitro models (Byatt et al 1991.…”