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• W2159886500 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/oGHR-ECDs 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. 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/oGHR-ECDs 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. growth hormone prolactin prolactin receptor growth hormone receptor extracellular domain placental lactogen human bovine ovine rat rabbit caprine polyacrylamide gel electrophoresis polymerase chain reaction lactogenic hormone response element with a Stat5 binding sequence Ruminant and other species' placentas synthesize and secrete unique proteins belonging to the growth hormone/prolactin (GH/PRL)1 family and are termed placental lactogens (PLs). Ovine (o) (1Martial J. Djiane J. Biochem. Biophys. Res. Commun. 1975; 65: 770-778Crossref PubMed Scopus (49) Google Scholar, 2Warren W.C. Liang R. Krivi G.G. Siegel N.R. Anthony R.V. J. Endocrinol. 1990; 126: 141-149Crossref PubMed Scopus (40) Google Scholar), bovine (b) (3Murthy G.S. Schellenberg C. Friesen H.G. Endocrinology. 1982; 111: 2117-2124Crossref PubMed Scopus (42) Google Scholar) and caprine (c) (4Curie J.C. Card C.E. Michel F.J. Ignotz G. Reprod. Fertil. 1990; 90: 25-26Crossref PubMed Scopus (24) Google Scholar) PLs were isolated from placentas and found to be 22–23-kDa proteins that are structurally closer to the respective PRLs than to GHs (5Byatt J.C. Warren W.C. Eppard P.J. Staten N.R. Krivi G.G. Collier R.J. J. Anim. Sci. 1992; 70: 1992-2911Crossref Scopus (59) Google Scholar). Recombinant oPL (6Colosi P. Thordarson G. Hellmiss R. Singh K. Forsyth I.A. Gluckman P.D. Wood W.I. Mol. Endocrinol. 1989; 3: 1462-1469Crossref PubMed Scopus (61) Google Scholar, 7Sakal E. Bignon C. Kantor A. Leibovitch H. Shamay A. Djiane J. Gertler A. J. Endocrinol. 1997; 152: 317-327Crossref PubMed Scopus (43) Google Scholar) and bPL (8.Krivi, G. G., Hauser, S. D., Stafford, J. M., Collier, J. M., Byatt, J. C., The Endocrine Society 71st Annual Meeting Abstracts, Abstr. 1523, 1991, The Endocrine Society Press, Bethesda, MD.Google Scholar), and recently cPL as well (9Sakal E. Bignon C. Chapnik-Cohen N. Daniel N. Paly J. Belair L. Djiane J. Gertler A. J. Endocrinol. 1998; 159: 509-518Crossref PubMed Scopus (26) Google Scholar), have been prepared, and the recombinant proteins can now be produced in amounts that allow in vivo studies. Cloning of cPL enabled us to compare its primary structure to that of oPL and bPL. The similarity between cPL and oPL exceeds the one between bPL and oPL or cPL. In contrast to these, the similarity between the corresponding GHs and PRLs in the three ruminant species is much greater (5Byatt J.C. Warren W.C. Eppard P.J. Staten N.R. Krivi G.G. Collier R.J. J. Anim. Sci. 1992; 70: 1992-2911Crossref Scopus (59) Google Scholar). It has been proposed that this finding suggests into possible different physiological roles that PLs may play in the three species, but this point has not yet been proven (5Byatt J.C. Warren W.C. Eppard P.J. Staten N.R. Krivi G.G. Collier R.J. J. Anim. Sci. 1992; 70: 1992-2911Crossref Scopus (59) Google Scholar). Recently, we have determined the three-dimensional structure of the 1:2 complex between oPL and the rPRLR-ECD, and have been able to identify the 25 residues of oPL that participate in site I of the hormone and 24 residues that participate in site II (10Christinger H.W. Elkins P.A. Sandowski Y. Sakal E. Gertler A. Kossiakoff A.A. De Vos A.M. Acta Crystallogr. D. 1998; 54: 1408-1411Crossref PubMed Google Scholar). This finding, along with our former direct and indirect experiments using recombinant extracellular domains (ECDs) of GH and PRL receptors, suggests that the initial step in PL signal transduction consists of dimerization of the respective receptor, as is well documented for GHs (11Wells J.A. De Vos A.M. Annu. Rev. Biochem. 1996; 65: 609-634Crossref PubMed Scopus (257) Google Scholar). One early observed, unique property of ruminant PLs is their ability to bind to both PRL and GH receptors, including receptors of hGH (for review, see Refs. 12Forsyth I.A. J. Dairy Sci. 1986; 69: 866-878Abstract Full Text PDF Scopus (139) Google Scholar, 13Anthony R.V. Liang R. Kayl E.P. Pratt S.L. J. Reprod. Fertil. Suppl. 1995; 49: 83-95PubMed Google Scholar, 14Anthony R.V. Pratt S.L. Liang R. Holland M.D. J. Anim. Sci. 1995; 73: 1861-1871Crossref PubMed Scopus (84) Google Scholar). Comparative binding studies of oPL and oGH to fetal liver microsomes, along with demonstration of oGHR mRNA in fetal liver, prompted several research groups to suggest that oGH and oPL bind to identical or at least related proteins (15Klempt M. Bingham B. Breier B.H. Baumach W.R. Gluckman P.D. Endocrinology. 1993; 132: 1071-1077Crossref PubMed Scopus (62) Google Scholar, 16Breier B.H. Funk B. Surus A. Ambler G.R. Wells C.A. Waters M.J. Gluckman P.D. Endocrinology. 1994; 135: 917-928Crossref Scopus (43) Google Scholar, 17Pratt S.L. Anthony R.V. Endocrinology. 1995; 136: 2150-2155Crossref PubMed Google Scholar). Using a similar approach, we have previously studied the biological activity of the three ruminant PLs in several in vitro bioassays, in which the signal was transduced through heterologous (mouse, rabbit, and human) GHRs (7Sakal E. Bignon C. Kantor A. Leibovitch H. Shamay A. Djiane J. Gertler A. J. Endocrinol. 1997; 152: 317-327Crossref PubMed Scopus (43) Google Scholar, 9Sakal E. Bignon C. Chapnik-Cohen N. Daniel N. Paly J. Belair L. Djiane J. Gertler A. J. Endocrinol. 1998; 159: 509-518Crossref PubMed Scopus (26) Google Scholar, 18Gertler A. Hauser S.D. Sakal E. Vashdi D. Staten N.R. Freeman J.J. Krivi G.G. J. Biol. Chem. 1992; 267: 12655-12659Abstract Full Text PDF PubMed Google Scholar, 19Vashdi D. Staten N.R. Sakal E. Krivi G.G. Gertler A. Endocrinology. 1995; 136: 1258-1266Crossref PubMed Scopus (15) Google Scholar, 20Vashdi-Elberg D. Staten N.R. Sakal E. McKinnie R.E. Djiane J. Krivi G.G. Gertler A. J. Biol. Chem. 1996; 271: 5558-5564Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 21Helman D. Staten N.R. Byatt J. Grosclaude J. McKinnie R.E. Djiane J. Gertler A. Endocrinology. 1997; 138: 4069-4080Crossref PubMed Scopus (13) Google Scholar, 22Helman D. Staten N.R. Grosclaude J. Daniel N. Neospoulous C. Djiane J. Gertler A. J. Biol. Chem. 1998; 273: 16067-16074Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In all cases the activity of bPL, oPL, or cPL was equal to that of oGH, bGH, or hGH despite some differences in affinity. Furthermore, mutagenesis of bPL allowed us to prepare several bPL analogues with the selectively reduced or abolished somatogenic activity, whereas the lactogenic activity (as judged by the Nb2 rat lymphoma cell proliferation bioassay) was not changed (20Vashdi-Elberg D. Staten N.R. Sakal E. McKinnie R.E. Djiane J. Krivi G.G. Gertler A. J. Biol. Chem. 1996; 271: 5558-5564Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 21Helman D. Staten N.R. Byatt J. Grosclaude J. McKinnie R.E. Djiane J. Gertler A. Endocrinology. 1997; 138: 4069-4080Crossref PubMed Scopus (13) Google Scholar, 22Helman D. Staten N.R. Grosclaude J. Daniel N. Neospoulous C. Djiane J. Gertler A. J. Biol. Chem. 1998; 273: 16067-16074Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). These experiments were paralleled by protein-interaction studies that showed that bPL, similarly to hGH, is capable of forming a 1:2 complex with h- and rbGHR-ECDs. In contrast to these results, Staten et al. (23Staten N.R. Byatt J.C. Krivi G.G. J. Biol. Chem. 1993; 268: 18467-18473Abstract Full Text PDF PubMed Google Scholar) reported that bPL interacts with bGHR-ECD in a 1:1 stoichiometry, whereas bGH forms a 1:2 complex. More recently, the same group briefly reported that bPL antagonized bGH action in Baf/3 cells stably transfected with bGHRs and was devoid of proliferative activity (24.Staten, N. R., Byatt, J. C., Krivi, G. G., The Endocrine Society 79th Annual Meeting Abstracts, Abstr. P2-212, 1997, The Endocrine Society Press, Bethesda, MD.Google Scholar). These reports prompted us to reexamine whether the somatogenic activity of ruminant PLs is relevant in homologous species. To answer this question, we developed an oGHR-mediated bioassay in 293 cells, prepared recombinant oGHR-ECD, and used them in the present study. Recombinant bPL, bPL G133R, bPL K73D, bPL T188D, oPL, oGH, and non-glycosylated human GHR-ECD were prepared as described previously (7Sakal E. Bignon C. Kantor A. Leibovitch H. Shamay A. Djiane J. Gertler A. J. Endocrinol. 1997; 152: 317-327Crossref PubMed Scopus (43) Google Scholar, 8.Krivi, G. G., Hauser, S. D., Stafford, J. M., Collier, J. M., Byatt, J. C., The Endocrine Society 71st Annual Meeting Abstracts, Abstr. 1523, 1991, The Endocrine Society Press, Bethesda, MD.Google Scholar, 18Gertler A. Hauser S.D. Sakal E. Vashdi D. Staten N.R. Freeman J.J. Krivi G.G. J. Biol. Chem. 1992; 267: 12655-12659Abstract Full Text PDF PubMed Google Scholar, 20Vashdi-Elberg D. Staten N.R. Sakal E. McKinnie R.E. Djiane J. Krivi G.G. Gertler A. J. Biol. Chem. 1996; 271: 5558-5564Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 21Helman D. Staten N.R. Byatt J. Grosclaude J. McKinnie R.E. Djiane J. Gertler A. Endocrinology. 1997; 138: 4069-4080Crossref PubMed Scopus (13) Google Scholar, 22Helman D. Staten N.R. Grosclaude J. Daniel N. Neospoulous C. Djiane J. Gertler A. J. Biol. Chem. 1998; 273: 16067-16074Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 25Tchelet A. Sakal E. Vogel T. Krivi G.G. Creely D. Gertler A. Pediatr. Adolesc. Endocrinol. 1993; 24: 114-126Google Scholar). Recombinant caprine (c) PL was recently prepared in our lab (9Sakal E. Bignon C. Chapnik-Cohen N. Daniel N. Paly J. Belair L. Djiane J. Gertler A. J. Endocrinol. 1998; 159: 509-518Crossref PubMed Scopus (26) Google Scholar). Rabbit (rb) non-glycosylated GHR-ECD was prepared in our laboratory, and its preparation will be described elsewhere. Carrier-free Na125I was purchased from NEN Life Science Products. Molecular weight markers for SDS-PAGE, RPMI 1640 medium, lysozyme, nalidixic acid, Triton X-100, bovine serum albumin (radioimmunoassay grade) were obtained from Sigma. SDS-PAGE reagents and protein assay kit were purchased from Bio-Rad. Fetal calf serum and horse serum were purchased from Labotal Co. (Jerusalem, Israel), and a SuperdexTM 75 HR 10/30 column and Q-Sepharose (fast flow) were obtained from Pharmacia LKB Biotechnology AB (Uppsala, Sweden). All other chemicals were of analytical grade. Synthetic gene fragment for preparation of oPL analogue T185D was constructed using polymerase chain reaction (PCR) technology. Oligonucleotides (primers) were used to generate a double-stranded DNA from a template, pET-8-oPL (7Sakal E. Bignon C. Kantor A. Leibovitch H. Shamay A. Djiane J. Gertler A. J. Endocrinol. 1997; 152: 317-327Crossref PubMed Scopus (43) Google Scholar), for subcloning. An NcoI site (underlined) was created with a forward primer at the 5′ end of the gene, which also added an initiator methionine codon immediately upstream to the first mature codon (alanine) (5′-GGAGATATACCATGGCACAGCATCCACC-3′) and aAflII site (underlined), was included close to mutation area at the 3′ end of the gene, with a reverse mutant primer (5′-GCACTTAAGTATCCGGAGGTAGTCGTAAATTTTAC-3′). The reverse mutant primer encoded the mutation of interest. The PCR reaction was conducted using Taq polymerase in a capillary PCR apparatus (Idaho Technology, Idaho Falls, ID), with the following program: 15 s × 94 °C, 25 cycles of (0 s × 94 °C, 0 s × 55 °C, 25 s × 72 °C), 15 s × 72 °C. The PCR product was gel-purified, subcloned into pGEM-T vector (Promega, Madison, WI), and transfected to JM-109 Escherichia colicells. The NcoI/AflII insert was isolated and ligated into the parental vector (pET8) encoding for the wild-type oPL (7Sakal E. Bignon C. Kantor A. Leibovitch H. Shamay A. Djiane J. Gertler A. J. Endocrinol. 1997; 152: 317-327Crossref PubMed Scopus (43) Google Scholar), from which the respective NcoI/AflII insert was removed. Subsequently, the pET8/oPL T185D cloning vector was isolated and used for transformation of BL21 E. coli cells. One of the isolated colonies that expressed the protein after induction by isopropyl-1-thio-β-d-galactopyranoside was selected for large scale preparation. Automatic DNA sequencing was performed to confirm the proper sequence. The oPL analogue expression vector was modified with the QuickchangeTM mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions, using two complementary primers: (5′-GGCCAAAGTACTTGTAGAACGTGTGGAAGTGATAC-3′) and (5′-GTATCACTTCACACGTTCTACAAGTACTTTGGCC-3′). These primers were designed to contain a specific restriction site (AflIII), still conserving the same amino acid sequence, for colony screening. The procedure included 12 PCR cycles and the use of Pfupolymerase enzyme for the reaction. The template used for mutant construction was wild-type oPL in pMON3922 (26Obukowicz M.G. Staten N.R. Krivi G.G. Appl. Environ. Microbiol. 1992; 58: 1511-1523Crossref PubMed Google Scholar). The mutated construct was then digested with DpnI restriction enzyme, which is specific to methylated and hemimethylated DNA (target sequence: 5′-Gm6ATC-3′), in order to digest the template and to select for mutation-containing synthesized DNA. The vector was then transfected into XL1-competent cells. Ten colonies were then screened for mutation, using the specific restriction site designed, and revealed 80% efficiency. Two colonies were sequenced and confirmed to contain the mutation and no undesired misincorporation of nucleotides. E. coli MON105 cells transformed with the expression plasmids containing the oPL G130R were incubated in 500 ml of Terrific Broth (TB) medium (27Tartof K.D. Hobbs C.A. Focus (BRL). 1987; 9: 12-20Google Scholar) by shaking at 200 rpm at 37 °C in 2-liter flasks to an A 600 of 0.9, after which nalidixic acid (25 mg/flask) was added. The cells were incubated for an additional period of 4 h, harvested by 5-min centrifugation at 10,000 × g, decanted, and then frozen at −20 °C. Over 95% of the expressed protein was found in the inclusion bodies, which were prepared as described previously for bPL (18Gertler A. Hauser S.D. Sakal E. Vashdi D. Staten N.R. Freeman J.J. Krivi G.G. J. Biol. Chem. 1992; 267: 12655-12659Abstract Full Text PDF PubMed Google Scholar). The inclusion body pellet obtained from 2.5 liters of bacterial culture was solubilized in 200 ml of 4.5 m urea buffered with 10 mm Tris base. The pH was increased to 11.3 with NaOH, cysteine was added to 0.1 mm, the clear solution was stirred at 4 °C for 1 h, diluted with two volumes of cold water, and dialyzed for an additional period of 48 h against 5 × 10 liters of 10 mm Tris-HCl, pH 9. The solution was subsequently loaded at 120 ml/h onto a Q-Sepharose column (2.6 × 7 cm), pre-equilibrated with 10 mm Tris-HCl, pH 9.0 at 4 °C. Elution was carried out using a discontinuous NaCl gradient in the same buffer at a rate of 120 ml/h, and 5-ml fractions were collected. Protein concentration was determined by absorbance at 280 nm, and monomer content by gel-filtration chromatography on a SuperdexTM 75 column. The oPL analogue T185D was expressed, according to the procedure described for the wild-type hormone (7Sakal E. Bignon C. Kantor A. Leibovitch H. Shamay A. Djiane J. Gertler A. J. Endocrinol. 1997; 152: 317-327Crossref PubMed Scopus (43) Google Scholar). Then it was refolded and purified as described above. Synthetic oligonucleotides (primers) were used to generate a double-stranded DNA from a template of full-size oGHR (35Adams T.E. Baker L. Fiddes R.J. Brandon M.R. Mol. Cell. Endocrinol. 1990; 73: 135-145Crossref PubMed Scopus (106) Google Scholar), in addition to restriction-enzyme sites for cloning. The forward primer 5′-GTGGCAGGCTCCACCATGGCTTTTTCTGGGAGTG-3′ encoded an NcoI restriction-enzyme site (underlined) and an initiator methionine codon immediately upstream to alanine codon and the oGHR-ECD. The reverse primer 5′- CCAAAGATAATAATTAAGAACCAAGCTTACTGGAAATC-3′ encoded theHindIII restriction site (underlined) and TAA termination codon immediately after the final codon (Gln).The PCR reaction was conducted using the Taq polymerase in capillary PCR apparatus (Idaho Technology), with the following program: 2 min × 94 °C, 30 cycles of (0 s × 94 °C, 0 s × 60 °C, 30 s × 72 °C), 2 min × 72 °C. The PCR product was cleaned using a Promega PCR cleaning kit (Promega, Madison, WI), digested with NcoI and HindIII restriction enzymes, and, after heat inactivation of the enzymes, ligated to parental vector pMON3401 (26Obukowicz M.G. Staten N.R. Krivi G.G. Appl. Environ. Microbiol. 1992; 58: 1511-1523Crossref PubMed Google Scholar) linearized with the same enzymes. The ligation product was transfected to JM-109 E. coli cells, prior to transformation of MON105 cells. Automatic DNA sequencing was performed to confirm the proper sequence. One of the expressing clones was chosen for large scale expression, which was performed as described above for oPL G130R analogue. Preparation of inclusion bodies and the refolding procedure was identical to that of oPL analogue G130R, except that after the solubilization in 4.5 m urea, the solution was stirred at 4 °C for 48 h, prior to dialysis against 10 mm Tris-HCl buffer pH 8.0 and subsequent purification on a Q-Sepharose column (2.6 × 7 cm), preequilibrated with the same buffer. The monomeric fraction was eluted with 150 mm NaCl at the same buffer, dialyzed, and lyophilized. Binding to soluble ovine, rabbit, and human GHR-ECDs was carried out as described previously (7Sakal E. Bignon C. Kantor A. Leibovitch H. Shamay A. Djiane J. Gertler A. J. Endocrinol. 1997; 152: 317-327Crossref PubMed Scopus (43) Google Scholar, 28Sandowski Y. Nagano M. Bignon C. Djiane J. Kelly P.A. Gertler A. Mol. Cell. Endocrinol. 1995; 115: 1-11Crossref PubMed Scopus (33) Google Scholar). The ligand was 125I-oGH or 125I-oPL, and the competitors were oGH, bPL, cPL, oPL, and oPL analogues. Iodination of oGH and oPL was performed according to the protocol described previously (29Gertler A. Ashkenazi A. Madar Z. Mol. Cell. Endocrinol. 1984; 34: 51-57Crossref PubMed Scopus (72) Google Scholar). High performance liquid chromatography gel-filtration chromatography on a SuperdexTM 75 HR 10/30 column was performed with 200-μl aliquots of Q-Sepharose-column-eluted fractions, freeze-dried samples dissolved in H2O, or complexes between the soluble recombinant GHR-ECDs and oGH, oPL, or oPL analogues, using methods described previously (7Sakal E. Bignon C. Kantor A. Leibovitch H. Shamay A. Djiane J. Gertler A. J. Endocrinol. 1997; 152: 317-327Crossref PubMed Scopus (43) Google Scholar, 30Bignon C. Sakal E. Belair L. Chapnik-Cohen N. Djiane J. Gertler A. J. Biol. Chem. 1994; 269: 3318-3324Abstract Full Text PDF PubMed Google Scholar). Twoin vitro bioassays, in which the signal was transduced through somatogenic receptors, were based on the proliferation of FDC-P1 cells transfected with rabbit (clone FDC-P1-3B9) or human (clone FDC-P1-9D11) GHRs (31Rowlinson S.W. Barnard R. Bastiras S. Robins A.J. Brinkworth R. Waters M.J. J. Biol. Chem. 1995; 270: 16833-16839Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 32Rowlinson S.W. Waters M.J. Lewis U.J. Barnard R. Endocrinology. 1996; 137: 90-95Crossref PubMed Scopus (55) Google Scholar) as described before (21Helman D. Staten N.R. Byatt J. Grosclaude J. McKinnie R.E. Djiane J. Gertler A. Endocrinology. 1997; 138: 4069-4080Crossref PubMed Scopus (13) Google Scholar). Cell growth was determined by counting the cells with a Coulter counter (Coulter Electronics Inc., Hialeah, FL), and the number of doublings was calculated as described previously (33Gertler A. Walker A. Friesen H.G. Endocrinology. 1985; 116: 1636-1644Crossref PubMed Scopus (86) Google Scholar). Two additional bioassays were carried out in a 293 cell line transiently transfected with hGHR or oGHR and co-transfected with a plasmid that carries the luciferase reporter gene under the control of a six-repeat sequence of LHRE (lactogenic hormone response element with a Stat5 binding sequence) fused to a minimal thymidine kinase promoter. The transfection and the bioassay were carried out as described previously (34Tchelet A. Vogel T. Helman D. Guy R. Neospoulous C. Goffin V. Djiane J. Gertler A. Mol. Cell. Endocrinol. 1997; 130: 141-151Crossref PubMed Scopus (10) Google Scholar). The vector encoding for full-size oGHR in SP72 vector was obtained from Dr. Tim Adams (35Adams T.E. Baker L. Fiddes R.J. Brandon M.R. Mol. Cell. Endocrinol. 1990; 73: 135-145Crossref PubMed Scopus (106) Google Scholar). It was first digested withXbaI at 37 °C for 1 h and then with EcoRI at room temperature for 5 min. The reaction products were then separated on 1.0% agarose gel, and the insert corresponding to ∼3200 bases was purified and ligated to pcDNA3 mammalian expression vector, linearized with XbaI and EcoRI. The ligated plasmid was propagated, isolated, and sequenced to ensure the proper ligation. An in vitro bioassay, in which the signal was transduced through lactogenic receptors, was performed in rat Nb2-11C lymphoma cell proliferation bioassay, in which the original protocol was slightly modified (33Gertler A. Walker A. Friesen H.G. Endocrinology. 1985; 116: 1636-1644Crossref PubMed Scopus (86) Google Scholar). The profile of oGHR-ECD elution from a Q-Sepharose column shows that over 60% of the protein was eluted with 0.15 m NaCl (data not shown). Every fifth tube was analyzed for monomer content, and fractions containing >98% pure monomer were pooled, dialyzed against NaHCO3(1:5 salt:protein ratio), and lyophilized. The overall yield was 110 mg of monomeric protein obtained from a 5-liter fermentation culture. This fraction was further used for binding and biological studies. Fractions eluted with 0.4 m NaCl consisted mainly of oligomers (data not shown). SDS-PAGE of the pooled monomer fraction, performed with and without β-mercaptoethanol according to Laemmli (36Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207521) Google Scholar), revealed only one band with a molecular mass of 28 kDa (data not shown). The oligomeric fraction eluted with 0.4 m NaCl has also yielded a main 28-kDa band, indicating that the oligomers were formed by a non-covalent interactions. The preparation of oPL analogues (oPL G130R and T185D) was carried out according to the protocol described for the wild-type recombinant oPL (7Sakal E. Bignon C. Kantor A. Leibovitch H. Shamay A. Djiane J. Gertler A. J. Endocrinol. 1997; 152: 317-327Crossref PubMed Scopus (43) Google Scholar). The monomeric fractions were eluted from the Q-Sepharose column developed with 10 mm Tris-HCl buffer at pH 9.0 by 0.05 m NaCl, dialyzed against NaHCO3 at a 4:1 protein:salt ratio and freeze-dried. The homogeneity of the purified proteins was also verified by SDS-PAGE under reducing and non-reducing conditions (data not shown). The biological activity of both analogues resulting from proper renaturation was further evidenced by their ability to stimulate the proliferation of the lactogenic receptor-mediated Nb2bioassay (data not shown) and to bind to human, rabbit, and ovine GHR-ECDs (Fig. 2) The relative activity of the G130R and T185D analogues in Nb2 bioassay as compared with wild-type oPL was, respectively, 2.5% and 87%. The stoichiometry of the interactions between soluble human, rabbit, and ovine GHR-ECDs and oPL, oPL T185D, oPL G130R and, in the latter case, also with oGH was studied by gel filtration. The complexes were prepared at a constant concentration (1.75 μm) of the hormones and variable concentrations (1.75–5.25 μm) of the respective receptor ECDs. Ovine PL formed a 1:2 complex with hGHR-ECDs, which was eluted at the retention time of 11.22 min (Fig.1 A), confirming the previous results (7Sakal E. Bignon C. Kantor A. Leibovitch H. Shamay A. Djiane J. Gertler A. J. Endocrinol. 1997; 152: 317-327Crossref PubMed Scopus (43) Google Scholar). At the oPL:hGH-ECD ratio of 1:3, an excess of hGHR-ECDs was observed. In contrast, both oPL T185D and G130R formed only 1:1 apparent complexes in both cases (Fig. 1 A). This conclusion was based on both a comparison of peak sizes, their retention times (11.94 and 12.44 min, respectively), and the fact that, at 1:2 analogue:hGHR-ECD ratios, an excess of the latter could be seen. The shape of the peaks of the oPL T185D:hGHR-ECD complexes and the fact that the retention time values shift forward by increasing the ECD:analogue ratio indicate that a very weak 1:1 complex that dissociated in the course of chromatography was likely formed. Similar results were also obtained with rbGHR-ECD (Fig. 1 B). In contrast, oPL was capable of forming only a 1:1 complex with oGHR-ECD, whereas with oGH, a clear 2:1 complex was detected (Fig.1 C). The oPL analogue G130R acted similarly to oPL. The results of the interaction of oPL T185D with oGHR-ECD do not, however, indicate formation of either 1:1 or 1:2 complexes, which likely results from the loss of binding capacity, as shown in the next section. As oPL is capable of binding to both homologous and heterologous somatogenic receptors, several binding assays were performed. The results of a comparative binding assay in which the ability of oPL, oPL G130K, and oPL T185D to compete with125I-oPL or 125I-oGH for binding to recombinant human, rabbit, and ovine GHR-ECDs is shown in Fig.2. The results (for binding of bPL and cPL to human and rabbit GHR-ECD, see our previous articles Refs. 7Sakal E. Bignon C. Kantor A. Leibovitch H. Shamay A. Djiane J. Gertler A. J. Endocrinol. 1997; 152: 317-327Crossref PubMed Scopus (43) Google Scholar, 9Sakal E. Bignon C. Chapnik-Cohen N. Daniel N. Paly J. Belair L. Djiane J. Gertler A. J. Endocrinol. 1998; 159: 509-518Crossref PubMed Scopus (26) Google Scholar, and 20Vashdi-Elberg D. Staten N.R. Sakal E. McKinnie R.E. Djiane J. Krivi G.G. Gertler A. J. Biol. Chem. 1996; 271: 5558-5564Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, 21Helman D. Staten N.R. Byatt J. Grosclaude J. McKinnie R.E. Djiane J. Gertler A. Endocrinology. 1997; 138: 4069-4080Crossref PubMed Scopus (13) Google Scholar, 22Helman D. Staten N.R. Grosclaude J. Daniel N. Neospoulous C. Djiane J. Gertler A. J. Biol. Chem. 1998; 273: 16067-16074Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) clearly demonstrate that all tested hormones (except oPL T185D) have an almost identical capacity to compete with the ligand for binding to h-, rb-, and oGHR-ECDs, respectively. The oPL analogue T185D exhibited 90 and >500 times lower competitive capacity in the binding to h- and rbGHR-ECDs, respectively, and no capacity at all in binding to oGHR-ECD. In all cases, results were calculated using both one-site and two-site analysis (37GraphPad PrismTMVersion 2.0. GraphPad Software Inc., San Diego1994Google Scholar). In the two-site analysis, calculations were based upon an assu" @default.
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• W2159886500 title "Ruminant Placental Lactogens Act as Antagonists to Homologous Growth Hormone Receptors and as Agonists to Human or Rabbit Growth Hormone Receptors" @default.
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