Bovine placental lactogen (bPL) was found to be as potent as human GH (hGH) in its ability to bind to soluble full-size recombinant hGH-binding protein (hGHBP) and to membrane-embedded hGH receptor in intact IM-9 human lymphocytes. bPL was also capable of forming a 1:2 complex with hGHBP, although the structure of this complex was probably more compact than that with hGH. Removal of 13 amino acids from the N-terminus of bPL did not affect its ability to bind to hGHBP or hGH receptors in intact IM-9 cells. Its ability to form a 1:2 complex with hGHBP was, however, impaired, unlike that of a corresponding analog in which an L28F mutation has been simultaneously introduced. Truncation of 17 amino acids decreased its affinity toward both hGHBP and GH receptors on intact IM-9 lymphocytes and in liver rat microsomal fraction and inhibited the formation of 1:2 complexes with hGHBP. Simultaneous L28F mutation did not affect affinity toward hGHBP, but increased affinity toward rat liver GH receptors and restored affinity toward membrane-embedded hGH receptors in IM-9 lymphocytes and the ability to form a 1:2 complex with hGHBP. Truncation of 20 amino acids further decreased affinity toward both hGHBP and receptors in intact IM-9 lymphocytes and completely abolished formation of a 1:2 complex with hGHBP. Both des-13-bPLs and bPL-des-17 (L28F) retained their full ability to stimulate insulin-like growth factor-I secretion by rat hepatocytes compared to that of bPL. The insulin-like growth factor-I stimulatory activities of bPL-des-17 and bPL-des-20, however, were decreased to 1-5%. These results indicate that the stoichiometry of 1:2 complex formation with hGHBP may be preserved despite decreased receptor binding affinity, but the lower affinity of the putative site 1 or site 2 of the analog may account for the decrease in biological activity. Furthermore, the ability or inability of bPL or its truncated analogs to form 1:2 complexes with soluble hGHBP cannot predict their somatogen receptor-mediated biological activity in rat hepatocytes.
Five recombinant analogues of bovine placental lactogen (bPL) ((bPL(S184H), bPL(S187A), bPL(S187F), bPL(T188F), bPL(T188F,I190F)) were prepared, expressed in Escherichia coli, and purified to homogeneity. Circular dichroism analysis revealed no or minor structural changes, except in bPL(T188F,I190F). Binding and biological activities of bPL(T188F,I190F) were almost completely abolished, whereas bPL analogues mutated at position 187 retained their full activity. Point mutation T188F resulted in selective modification; binding to somatogenic receptors, their extracellular domains (ECDs), and to bPLR in the endometrium as well as somatogenic receptor-mediated biological activities were reduced or abolished, whereas binding to lactogenic receptors, their ECDs, and subsequent biological activity was fully or almost fully retained. This selective modification most likely results from a steric hindrance induced by a bulky Phe-188 chain of bPL which interacts with the Arg-43 of the human or Leu-43 of the non-human GHRs. Point mutation S184H abolished the interaction with hGHR, most likely due to the unfavorable charge-charge interaction, possibly accompanied by steric hindrance between Arg-43 of the receptor and the newly introduced His-184 and possible interference with the putative interaction between the alkyl portion of Thr-188 and Lys-185 of bPL with Trp-104 of hGHR. In contrast, bPL(S184H) retained its capacity to interact with nonhuman GHRs. Decrease in the biological activity of bPL(S184H) was also observed in two lactogenic receptor-mediated bioassays most likely due to the elimination of the intermolecular hydrogen bond of Ser-184 with a side chain of Tyr-127, which appears in all lactogenic receptors. Bovine placental lactogen (bPL)1 has been purified from term placental homogenates (1) and from isolated secretory granules obtained from binucleate cells of fetal cotyledon (2). The native 31 to 33 kDa bPL has at least five isoelectric variants which are in part due to heterogeneity of the attached oligosaccharides, and to as yet unidentified modifications. The gene for bPL has been cloned and expressed with high efficiency in Escherichia coli and the recombinant bPL has been purified to homogeneity (3). The predicted mature bPL has 200 residues and the primary sequence exhibits 50% and 23% homology to bovine prolactin (bPRL) and growth hormone (bGH), respectively (3). In comparison with bGH and hGH, bPL has 12-13 additional amino acids at the N-terminal portion of the molecule and, in common with mammalian PRLs, it has a third disulfide bond located in this N-terminal region. Deglycosylation of native bPL had no effect on PRL-like mitogenic activity in an Nb 2 lymphoma cell proliferation assay in which bPL was equally potent to human (h)GH, bPRL, and ovine (o)PRL, and exhibited only slightly reduced binding to bGH receptors (4). Recombinant bPL was also equally potent to hGH or oGH in somatogenic receptor-mediated 3T3-L1 or 3T3-F442A preadipocyte bioassays (5, 6). However, in a homologous lactogenic receptorme...
Bovine placental lactogen (bPL) was found to be as potent as human GH (hGH) in its ability to bind to soluble full-size recombinant hGH-binding protein (hGHBP) and to membrane-embedded hGH receptor in intact IM-9 human lymphocytes. bPL was also capable of forming a 1:2 complex with hGHBP, although the structure of this complex was probably more compact than that with hGH. Removal of 13 amino acids from the N-terminus of bPL did not affect its ability to bind to hGHBP or hGH receptors in intact IM-9 cells. Its ability to form a 1:2 complex with hGHBP was, however, impaired, unlike that of a corresponding analog in which an L28F mutation has been simultaneously introduced. Truncation of 17 amino acids decreased its affinity toward both hGHBP and GH receptors on intact IM-9 lymphocytes and in liver rat microsomal fraction and inhibited the formation of 1:2 complexes with hGHBP. Simultaneous L28F mutation did not affect affinity toward hGHBP, but increased affinity toward rat liver GH receptors and restored affinity toward membrane-embedded hGH receptors in IM-9 lymphocytes and the ability to form a 1:2 complex with hGHBP. Truncation of 20 amino acids further decreased affinity toward both hGHBP and receptors in intact IM-9 lymphocytes and completely abolished formation of a 1:2 complex with hGHBP. Both des-13-bPLs and bPL-des-17 (L28F) retained their full ability to stimulate insulin-like growth factor-I secretion by rat hepatocytes compared to that of bPL. The insulin-like growth factor-I stimulatory activities of bPL-des-17 and bPL-des-20, however, were decreased to 1-5%. These results indicate that the stoichiometry of 1:2 complex formation with hGHBP may be preserved despite decreased receptor binding affinity, but the lower affinity of the putative site 1 or site 2 of the analog may account for the decrease in biological activity. Furthermore, the ability or inability of bPL or its truncated analogs to form 1:2 complexes with soluble hGHBP cannot predict their somatogen receptor-mediated biological activity in rat hepatocytes.
Lactogenic hormone-dependent Nb2-11C cells proliferate in response to prolactin (PRL) or human growth hormone (hGH). We have investigated the activation of p21( ras ) and mitogen-activated protein kinase (MAP-kinase) by hGH in lactogen-dependent Nb2-11C and in autonomous hormone-independent Nb2-SP rat lymphoma cells. Exposure of Nb2-11C cells to hGH resulted in a dose-dependent activation of p21( ras ) and of MAP-kinase. Activation occurs at physiological hGH concentration and with a rapid onset (∼1 min) reaching maximal level at 10-20 min. In contrast, in Nb2-SP autonomous lactogen-independent cells, p21( ras ) and MAP-kinase are constitutively activated and a challenge with lactogenic hormone had a modest additional activating effect. TPA, an activator of protein kinase C, enhanced p21( ras ) and MAP-kinase activity in Nb2-11C cells but failed to induce proliferation. The mechanism of activation of p21( ras ) in Nb2-11C cells by lactogenic hormones involves both an increased binding of guanine nucleotides to p21( ras ) as well as an increase in GTP/GDP+GTP ratio. In summary, we have demonstrated here that activation of the p21( ras )/MAP-kinase pathway follows PRL receptor activation but is not sufficient for the lactogenic hormone-dependent mitogenesis.
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