Antagonistic Properties of Human Prolactin Analogs That Show Paradoxical Agonistic Activity in the Nb2 Bioassay

Abstract

Based on the assumption that the prolactin receptor (PRLR) is activated by PRL-induced sequential dimerization, potential human PRL (hPRL) antagonists were designed that sterically interfere with binding site 2. We previously reported the unexpected agonistic properties of these hPRL analogs in the rat Nb2 bioassay (Goffin, V., Struman, I., Mainfroid, V., Kinet, S., and Martial, J. A. (1994) J. Biol. Chem. 269, 32598-32606). In order to investigate whether such paradoxical agonistic behavior might result from characteristic features of the Nb2 assay (e.g. species specificity), we transfected in the same cell system the cDNA encoding the PRLR from rat or human species along with reporter genes containing PRL-responsive DNA sequences. We characterized the agonistic, self-antagonistic and/or antagonistic effects of wild type rat PRL, wild type hPRL, and three hPRL analogs, mutated either at binding site 1 or at binding site 2. Our results clearly show that the agonistic/antagonistic properties of PRLs are species-specific. We thus propose different models of receptor activation, depending on the relative affinities of each hormonal binding site, which is directed by species specificity. Finally, this is the first report of hPRL binding site 2 analogs showing antagonistic properties on human and, to a lesser extent, rat receptors. Based on the assumption that the prolactin receptor (PRLR) is activated by PRL-induced sequential dimerization, potential human PRL (hPRL) antagonists were designed that sterically interfere with binding site 2. We previously reported the unexpected agonistic properties of these hPRL analogs in the rat Nb2 bioassay (Goffin, V., Struman, I., Mainfroid, V., Kinet, S., and Martial, J. A. (1994) J. Biol. Chem. 269, 32598-32606). In order to investigate whether such paradoxical agonistic behavior might result from characteristic features of the Nb2 assay (e.g. species specificity), we transfected in the same cell system the cDNA encoding the PRLR from rat or human species along with reporter genes containing PRL-responsive DNA sequences. We characterized the agonistic, self-antagonistic and/or antagonistic effects of wild type rat PRL, wild type hPRL, and three hPRL analogs, mutated either at binding site 1 or at binding site 2. Our results clearly show that the agonistic/antagonistic properties of PRLs are species-specific. We thus propose different models of receptor activation, depending on the relative affinities of each hormonal binding site, which is directed by species specificity. Finally, this is the first report of hPRL binding site 2 analogs showing antagonistic properties on human and, to a lesser extent, rat receptors. INTRODUCTIONProlactin (PRL) 1The abbreviations used are: PRLprolactinhPRLhuman PRLrPRLrat PRLPRLRPRL receptorGHgrowth hormonehGHhuman GHGHRgrowth hormone receptorhGHRhuman GHRWTwild typehGHbphGH binding proteinPRLbpPRL binding proteinhPRLbphPRL binding proteinPLplacental lactogenLHRElactogenic hormone response element. and growth hormone (GH) are homologous hormones primarily secreted by the anterior pituitary in all vertebrates (for review, see 1Miller W.L. Eberhardt N.L. Endocr. Rev. 1983; 4: 97-129Crossref PubMed Scopus (292) Google Scholar). They are involved in a wide spectrum of biological activities, mainly related to lactation, reproduction, and immunomodulation for PRL and growth and morphogenesis for GH (for review, see 2Meites J. Hoshino K. Prolactin Gene Family and Its Receptors. Excerpta Medica, Amsterdam1988: 123Google Scholar and 3Lewis U.J. Trends Endocrinol. Metab. 1992; 3: 117-121Abstract Full Text PDF PubMed Scopus (66) Google Scholar). These bioactivities are mediated by homologous membrane receptors, the PRL receptor (PRLR), also known as the lactogen receptor, and the GH receptor (GHR), also referred to as the somatogen receptor. Both belong to the cytokine receptor superfamily (for reviews, see 4Kelly P.A. Djiane J. Postel-Vinay M.-C. Edery M. Endocr. Rev. 1991; 12: 235-251Crossref PubMed Scopus (658) Google Scholar, 5Kelly P.A. Ali S. Rozakis M. Goujon L. Nagano M. Pellegrini I. Gould D. Djiane J. Edery M. Finidori J. Postel-Vinay M.-C. Recent. Prog. Horm. Res. 1993; 48: 123-164Crossref PubMed Google Scholar, 6Horseman N.D. Yu-Lee L.Y. Endocr. Rev. 1994; 15: 627-649Crossref PubMed Scopus (194) Google Scholar).Activation of the GHR and the PRLR is assumed to occur upon ligand-induced dimerization. For the GHR, mutational (7Cunningham B.C. Ultsch M. de Vos A.M. Mulkerrin M.G. Clauser K.R. Wells J.A. Science. 1991; 254: 821-825Crossref PubMed Scopus (780) Google Scholar) and structural (8de Vos A.M. Ultsch M. Kossiakoff A.A. Science. 1992; 255: 306-312Crossref PubMed Scopus (2014) Google Scholar) studies have demonstrated that a single human GH (hGH) molecule binds to two molecules of hGH binding protein (hGHbp) through two regions referred to as binding sites 1 and 2. Binding site 1 is delimited by a pocket encompassed within helix 1, helix 4, and loop 1, whereas binding site 2 involves a cleft delimited by the opposite side of helix 1 and helix 3 as well as few residues of the N terminus. A sequential dimerization model for membrane-bound receptor has been proposed (9Fuh G. Cunningham B.C. Fukunaga R. Nagata S. Goeddel D.V. Wells J.A. Science. 1992; 256: 1677-1680Crossref PubMed Scopus (571) Google Scholar). In a first step, one hGH molecule binds to one hGHR molecule through its site 1 to form a 1:1 ligand-receptor complex that remains inactive. In a second step, the hGH involved in this intermediate 1:1 complex binds, through its site 2, to a second hGHR molecule, and an active 1:2 hormone-receptor complex results. For the PRLR, a similar mechanism of receptor activation is anticipated, since we have identified on hPRL two binding sites involving regions of the protein topologically equivalent to those forming the binding sites of hGH (10Goffin V. Norman M. Martial J.A. Mol. Endocrinol. 1992; 6: 1381-1392PubMed Google Scholar, 11Goffin V. Struman I. Goormaghtigh E. Martial J.A. Eur. J. Biochem. 1993; 214: 483-490Crossref PubMed Scopus (15) Google Scholar, 12Goffin V. Struman I. Mainfroid V. Kinet S. Martial J.A. J. Biol. Chem. 1994; 269: 32598-32606Abstract Full Text PDF PubMed Google Scholar, 13Goffin V. Martial J.A. Summers N.L. Protein Eng. 1995; 12: 1215-1231Crossref Scopus (41) Google Scholar, 14Kinet S. Goffin V. Mainfroid V. Martial J.A. J. Biol. Chem. 1996; 271: 14353-14360Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Moreover, the hGH-hGHbp and hGH-hPRLbp 1:1 complexes were found to have very similar overall structures (8de Vos A.M. Ultsch M. Kossiakoff A.A. Science. 1992; 255: 306-312Crossref PubMed Scopus (2014) Google Scholar, 15Kossiakoff A.A. Somers W. Ultsch M. Andow K. Muller Y.A. de Vos A.M. Protein Sci. 1994; 3: 1697-1705Crossref PubMed Scopus (78) Google Scholar, 16Somers W. Ultsch M. de Vos A.M. Kossiakoff A.A. Nature. 1994; 372: 478-481Crossref PubMed Scopus (356) Google Scholar), strengthening analogies between these interactions involving these homologous hormones and receptors.Based on the critical role of the cleft at binding site 2, which buries two Trp residues of the receptor upon binding (8de Vos A.M. Ultsch M. Kossiakoff A.A. Science. 1992; 255: 306-312Crossref PubMed Scopus (2014) Google Scholar, 17Bass S.H. Mulkerrin M.G. Wells J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4498-4502Crossref PubMed Scopus (253) Google Scholar, and 18Clarckson T. Wells J.A. Science. 1995; 267: 383-386Crossref PubMed Scopus (1767) Google Scholar; for review, see 15Kossiakoff A.A. Somers W. Ultsch M. Andow K. Muller Y.A. de Vos A.M. Protein Sci. 1994; 3: 1697-1705Crossref PubMed Scopus (78) Google Scholar), a GHR antagonist has been designed. By replacing the hydrogen side chain of Gly120 (helix 3) in hGH with the much larger side chain of an Arg (so-called G120R hGH analog), the cleft within the helix 1/helix 3 interface is sterically hindered, and this analog, unable to induce receptor dimerization, fails to exhibit any agonistic activity (9Fuh G. Cunningham B.C. Fukunaga R. Nagata S. Goeddel D.V. Wells J.A. Science. 1992; 256: 1677-1680Crossref PubMed Scopus (571) Google Scholar). Conversely, it acts at high concentrations as an antagonist of WT hGH by blocking hormone-receptor complexes in the inactive 1:1 stoichiometry (9Fuh G. Cunningham B.C. Fukunaga R. Nagata S. Goeddel D.V. Wells J.A. Science. 1992; 256: 1677-1680Crossref PubMed Scopus (571) Google Scholar). The conservation of the helix 3 glycine within the PRL/GH family (19Nicoll C.S. Mayer G.L. Russell S.M. Endocr. Rev. 1986; 7: 169-203Crossref PubMed Scopus (324) Google Scholar) argues for a conserved functional role of this residue in maintaining the cleft at binding site 2. In agreement, the antagonistic properties of such GH analogs, first assessed on the GHR from humans (9Fuh G. Cunningham B.C. Fukunaga R. Nagata S. Goeddel D.V. Wells J.A. Science. 1992; 256: 1677-1680Crossref PubMed Scopus (571) Google Scholar), were then observed on rat and mice GHR (20Ilondo M.M. Damholt A.B. Cunningham B.C. Wells J.A. De Meyts P. Shymko R.M. Endocrinology. 1994; 134: 2397-2403Crossref PubMed Scopus (103) Google Scholar, 21Chen W.Y. Wight D.C. Mehta B.V. Wagner T.E. Kopchick J.J. Mol. Endocrinol. 1991; 5: 1845-1852Crossref PubMed Scopus (145) Google Scholar) as well as on human and rat PRLR (22Fuh G. Colosi P. Wood W.I. Wells J.A. J. Biol. Chem. 1993; 268: 5376-5381Abstract Full Text PDF PubMed Google Scholar). In contrast, we recently showed that the G129R hPRL analog (homologous to G120R hGH) failed to antagonize hPRL-induced proliferation of rat Nb2 cells (12Goffin V. Struman I. Mainfroid V. Kinet S. Martial J.A. J. Biol. Chem. 1994; 269: 32598-32606Abstract Full Text PDF PubMed Google Scholar). These observations led us to postulate that the mechanism of PRLR dimerization shows some differences depending on the intrinsic properties of PRL versus GH (12Goffin V. Struman I. Mainfroid V. Kinet S. Martial J.A. J. Biol. Chem. 1994; 269: 32598-32606Abstract Full Text PDF PubMed Google Scholar).Within the past 2 years, however, conflicting data have been reported that question the ubiquitous antagonistic properties of GH analogs mutated at binding site 2. First, a recent report demonstrates that the G120R hGH analog actually acts as an agonist on rat Nb2 cells in the absence of zinc ions (23Dattani M.T. Hindmarsh P.C. Brook C.G.D. Robinson I.C.A. Kopchick J.J. Marshall N.J. J. Biol. Chem. 1995; 270: 9222-9226Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Second, Mode et al. (24Mode A. Tollet P. Wlees T. Carmignac D.F. Clarck R.G. Chen W.Y. Kopchick J.J. Robinson I.C.A.F. Endocrinology. 1996; 137: 447-454Crossref PubMed Google Scholar) failed to observe any GHR antagonism of the hGH G120R analog when infused in rats but moreover found that this variant displayed agonistic activity via the PRLR. Third, although the I4A hGH analog is poorly able to dimerize the hGHbp in vitro (7Cunningham B.C. Ultsch M. de Vos A.M. Mulkerrin M.G. Clauser K.R. Wells J.A. Science. 1991; 254: 821-825Crossref PubMed Scopus (780) Google Scholar), it acts as a perfect GH agonist in transgenic mice (25Chen W.Y. Chen N.Y. Yun J. Wagner T.E. Kopchick J.J. J. Biol. Chem. 1994; 269: 15892-15897Abstract Full Text PDF PubMed Google Scholar, 26Chen W.Y. Chen N.Y. Yun J. Wight D.C. Wang X.Z. Wagner T.E. Kopchick J.J. Mol. Endocrinol. 1995; 9: 292-302Crossref PubMed Google Scholar). Accordingly, a hGH variant lacking the first seven N-terminal residues displayed full agonistic activity in the rat Nb2 assay (27Binder L. Gertler A. Elberg G. Guy R. Vogel T. Mol. Endocrinol. 1990; 4: 1060-1068Crossref PubMed Scopus (21) Google Scholar). Taken together, these data suggest that the antagonistic properties of PRL/GH analogs mutated at binding site 2 might not only depend on the properties of the hormones themselves, as usually expected, but also on some features (e.g. species specificity, sensitivity) of the bioassays used for their functional characterization.Studies on lactogens are most often performed using the rat Nb2 cell proliferation bioassay (10Goffin V. Norman M. Martial J.A. Mol. Endocrinol. 1992; 6: 1381-1392PubMed Google Scholar, 11Goffin V. Struman I. Goormaghtigh E. Martial J.A. Eur. J. Biochem. 1993; 214: 483-490Crossref PubMed Scopus (15) Google Scholar, 12Goffin V. Struman I. Mainfroid V. Kinet S. Martial J.A. J. Biol. Chem. 1994; 269: 32598-32606Abstract Full Text PDF PubMed Google Scholar, 14Kinet S. Goffin V. Mainfroid V. Martial J.A. J. Biol. Chem. 1996; 271: 14353-14360Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 27Binder L. Gertler A. Elberg G. Guy R. Vogel T. Mol. Endocrinol. 1990; 4: 1060-1068Crossref PubMed Scopus (21) Google Scholar, 28Gout P.W. Beer C.T. Noble R.L. Cancer Res. 1980; 40: 2433-2436PubMed Google Scholar, 29Tanaka T. Shiu R.P.C. Gout P.W. Beer C.T. Noble R.L. Friesen H.G. J. Clin. Endocrinol. & Metab. 1980; 51: 1058-1063Crossref PubMed Scopus (565) Google Scholar, 30Luck D.N. Gout P.W. Kelsay K. Atkinson T. Beer C.T. Smith M. Mol. Endocrinol. 1990; 4: 1011-1016Crossref PubMed Scopus (18) Google Scholar, 31Luck D.N. Huyer M. Gout P.W. Beer C.T. Smith M. Mol. Endocrinol. 1991; 5: 1880-1886Crossref PubMed Scopus (27) Google Scholar). In order to investigate if the use of this bioassay might be misleading for assessing antagonistic properties of lactogen analogs, such as G129R hPRL (12Goffin V. Struman I. Mainfroid V. Kinet S. Martial J.A. J. Biol. Chem. 1994; 269: 32598-32606Abstract Full Text PDF PubMed Google Scholar), we have analyzed several PRLs, wild type (WT) or mutated, using an in vitro bioassay in which the transcriptional activation of PRL-responsive reporter genes is mediated by exogenous PRLR from rat or human species, which allows their comparison in a strictly identical cell system. Our data clearly show that (i) the rat PRLR displays a higher affinity than human PRLR for binding site 2 of PRLs, (ii) consequently, the agonistic and antagonistic properties of a given analog are driven by species specific features, and (iii) the hPRL analog G129R displays antagonistic properties on PRLR from both species, in to we previously observed using the Nb2 proliferation bioassay (12Goffin V. Struman I. Mainfroid V. Kinet S. Martial J.A. J. Biol. Chem. 1994; 269: 32598-32606Abstract Full Text PDF PubMed Google Scholar). INTRODUCTIONProlactin (PRL) 1The abbreviations used are: PRLprolactinhPRLhuman PRLrPRLrat PRLPRLRPRL receptorGHgrowth hormonehGHhuman GHGHRgrowth hormone receptorhGHRhuman GHRWTwild typehGHbphGH binding proteinPRLbpPRL binding proteinhPRLbphPRL binding proteinPLplacental lactogenLHRElactogenic hormone response element. and growth hormone (GH) are homologous hormones primarily secreted by the anterior pituitary in all vertebrates (for review, see 1Miller W.L. Eberhardt N.L. Endocr. Rev. 1983; 4: 97-129Crossref PubMed Scopus (292) Google Scholar). They are involved in a wide spectrum of biological activities, mainly related to lactation, reproduction, and immunomodulation for PRL and growth and morphogenesis for GH (for review, see 2Meites J. Hoshino K. Prolactin Gene Family and Its Receptors. Excerpta Medica, Amsterdam1988: 123Google Scholar and 3Lewis U.J. Trends Endocrinol. Metab. 1992; 3: 117-121Abstract Full Text PDF PubMed Scopus (66) Google Scholar). These bioactivities are mediated by homologous membrane receptors, the PRL receptor (PRLR), also known as the lactogen receptor, and the GH receptor (GHR), also referred to as the somatogen receptor. Both belong to the cytokine receptor superfamily (for reviews, see 4Kelly P.A. Djiane J. Postel-Vinay M.-C. Edery M. Endocr. Rev. 1991; 12: 235-251Crossref PubMed Scopus (658) Google Scholar, 5Kelly P.A. Ali S. Rozakis M. Goujon L. Nagano M. Pellegrini I. Gould D. Djiane J. Edery M. Finidori J. Postel-Vinay M.-C. Recent. Prog. Horm. Res. 1993; 48: 123-164Crossref PubMed Google Scholar, 6Horseman N.D. Yu-Lee L.Y. Endocr. Rev. 1994; 15: 627-649Crossref PubMed Scopus (194) Google Scholar).Activation of the GHR and the PRLR is assumed to occur upon ligand-induced dimerization. For the GHR, mutational (7Cunningham B.C. Ultsch M. de Vos A.M. Mulkerrin M.G. Clauser K.R. Wells J.A. Science. 1991; 254: 821-825Crossref PubMed Scopus (780) Google Scholar) and structural (8de Vos A.M. Ultsch M. Kossiakoff A.A. Science. 1992; 255: 306-312Crossref PubMed Scopus (2014) Google Scholar) studies have demonstrated that a single human GH (hGH) molecule binds to two molecules of hGH binding protein (hGHbp) through two regions referred to as binding sites 1 and 2. Binding site 1 is delimited by a pocket encompassed within helix 1, helix 4, and loop 1, whereas binding site 2 involves a cleft delimited by the opposite side of helix 1 and helix 3 as well as few residues of the N terminus. A sequential dimerization model for membrane-bound receptor has been proposed (9Fuh G. Cunningham B.C. Fukunaga R. Nagata S. Goeddel D.V. Wells J.A. Science. 1992; 256: 1677-1680Crossref PubMed Scopus (571) Google Scholar). In a first step, one hGH molecule binds to one hGHR molecule through its site 1 to form a 1:1 ligand-receptor complex that remains inactive. In a second step, the hGH involved in this intermediate 1:1 complex binds, through its site 2, to a second hGHR molecule, and an active 1:2 hormone-receptor complex results. For the PRLR, a similar mechanism of receptor activation is anticipated, since we have identified on hPRL two binding sites involving regions of the protein topologically equivalent to those forming the binding sites of hGH (10Goffin V. Norman M. Martial J.A. Mol. Endocrinol. 1992; 6: 1381-1392PubMed Google Scholar, 11Goffin V. Struman I. Goormaghtigh E. Martial J.A. Eur. J. Biochem. 1993; 214: 483-490Crossref PubMed Scopus (15) Google Scholar, 12Goffin V. Struman I. Mainfroid V. Kinet S. Martial J.A. J. Biol. Chem. 1994; 269: 32598-32606Abstract Full Text PDF PubMed Google Scholar, 13Goffin V. Martial J.A. Summers N.L. Protein Eng. 1995; 12: 1215-1231Crossref Scopus (41) Google Scholar, 14Kinet S. Goffin V. Mainfroid V. Martial J.A. J. Biol. Chem. 1996; 271: 14353-14360Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Moreover, the hGH-hGHbp and hGH-hPRLbp 1:1 complexes were found to have very similar overall structures (8de Vos A.M. Ultsch M. Kossiakoff A.A. Science. 1992; 255: 306-312Crossref PubMed Scopus (2014) Google Scholar, 15Kossiakoff A.A. Somers W. Ultsch M. Andow K. Muller Y.A. de Vos A.M. Protein Sci. 1994; 3: 1697-1705Crossref PubMed Scopus (78) Google Scholar, 16Somers W. Ultsch M. de Vos A.M. Kossiakoff A.A. Nature. 1994; 372: 478-481Crossref PubMed Scopus (356) Google Scholar), strengthening analogies between these interactions involving these homologous hormones and receptors.Based on the critical role of the cleft at binding site 2, which buries two Trp residues of the receptor upon binding (8de Vos A.M. Ultsch M. Kossiakoff A.A. Science. 1992; 255: 306-312Crossref PubMed Scopus (2014) Google Scholar, 17Bass S.H. Mulkerrin M.G. Wells J.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4498-4502Crossref PubMed Scopus (253) Google Scholar, and 18Clarckson T. Wells J.A. Science. 1995; 267: 383-386Crossref PubMed Scopus (1767) Google Scholar; for review, see 15Kossiakoff A.A. Somers W. Ultsch M. Andow K. Muller Y.A. de Vos A.M. Protein Sci. 1994; 3: 1697-1705Crossref PubMed Scopus (78) Google Scholar), a GHR antagonist has been designed. By replacing the hydrogen side chain of Gly120 (helix 3) in hGH with the much larger side chain of an Arg (so-called G120R hGH analog), the cleft within the helix 1/helix 3 interface is sterically hindered, and this analog, unable to induce receptor dimerization, fails to exhibit any agonistic activity (9Fuh G. Cunningham B.C. Fukunaga R. Nagata S. Goeddel D.V. Wells J.A. Science. 1992; 256: 1677-1680Crossref PubMed Scopus (571) Google Scholar). Conversely, it acts at high concentrations as an antagonist of WT hGH by blocking hormone-receptor complexes in the inactive 1:1 stoichiometry (9Fuh G. Cunningham B.C. Fukunaga R. Nagata S. Goeddel D.V. Wells J.A. Science. 1992; 256: 1677-1680Crossref PubMed Scopus (571) Google Scholar). The conservation of the helix 3 glycine within the PRL/GH family (19Nicoll C.S. Mayer G.L. Russell S.M. Endocr. Rev. 1986; 7: 169-203Crossref PubMed Scopus (324) Google Scholar) argues for a conserved functional role of this residue in maintaining the cleft at binding site 2. In agreement, the antagonistic properties of such GH analogs, first assessed on the GHR from humans (9Fuh G. Cunningham B.C. Fukunaga R. Nagata S. Goeddel D.V. Wells J.A. Science. 1992; 256: 1677-1680Crossref PubMed Scopus (571) Google Scholar), were then observed on rat and mice GHR (20Ilondo M.M. Damholt A.B. Cunningham B.C. Wells J.A. De Meyts P. Shymko R.M. Endocrinology. 1994; 134: 2397-2403Crossref PubMed Scopus (103) Google Scholar, 21Chen W.Y. Wight D.C. Mehta B.V. Wagner T.E. Kopchick J.J. Mol. Endocrinol. 1991; 5: 1845-1852Crossref PubMed Scopus (145) Google Scholar) as well as on human and rat PRLR (22Fuh G. Colosi P. Wood W.I. Wells J.A. J. Biol. Chem. 1993; 268: 5376-5381Abstract Full Text PDF PubMed Google Scholar). In contrast, we recently showed that the G129R hPRL analog (homologous to G120R hGH) failed to antagonize hPRL-induced proliferation of rat Nb2 cells (12Goffin V. Struman I. Mainfroid V. Kinet S. Martial J.A. J. Biol. Chem. 1994; 269: 32598-32606Abstract Full Text PDF PubMed Google Scholar). These observations led us to postulate that the mechanism of PRLR dimerization shows some differences depending on the intrinsic properties of PRL versus GH (12Goffin V. Struman I. Mainfroid V. Kinet S. Martial J.A. J. Biol. Chem. 1994; 269: 32598-32606Abstract Full Text PDF PubMed Google Scholar).Within the past 2 years, however, conflicting data have been reported that question the ubiquitous antagonistic properties of GH analogs mutated at binding site 2. First, a recent report demonstrates that the G120R hGH analog actually acts as an agonist on rat Nb2 cells in the absence of zinc ions (23Dattani M.T. Hindmarsh P.C. Brook C.G.D. Robinson I.C.A. Kopchick J.J. Marshall N.J. J. Biol. Chem. 1995; 270: 9222-9226Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Second, Mode et al. (24Mode A. Tollet P. Wlees T. Carmignac D.F. Clarck R.G. Chen W.Y. Kopchick J.J. Robinson I.C.A.F. Endocrinology. 1996; 137: 447-454Crossref PubMed Google Scholar) failed to observe any GHR antagonism of the hGH G120R analog when infused in rats but moreover found that this variant displayed agonistic activity via the PRLR. Third, although the I4A hGH analog is poorly able to dimerize the hGHbp in vitro (7Cunningham B.C. Ultsch M. de Vos A.M. Mulkerrin M.G. Clauser K.R. Wells J.A. Science. 1991; 254: 821-825Crossref PubMed Scopus (780) Google Scholar), it acts as a perfect GH agonist in transgenic mice (25Chen W.Y. Chen N.Y. Yun J. Wagner T.E. Kopchick J.J. J. Biol. Chem. 1994; 269: 15892-15897Abstract Full Text PDF PubMed Google Scholar, 26Chen W.Y. Chen N.Y. Yun J. Wight D.C. Wang X.Z. Wagner T.E. Kopchick J.J. Mol. Endocrinol. 1995; 9: 292-302Crossref PubMed Google Scholar). Accordingly, a hGH variant lacking the first seven N-terminal residues displayed full agonistic activity in the rat Nb2 assay (27Binder L. Gertler A. Elberg G. Guy R. Vogel T. Mol. Endocrinol. 1990; 4: 1060-1068Crossref PubMed Scopus (21) Google Scholar). Taken together, these data suggest that the antagonistic properties of PRL/GH analogs mutated at binding site 2 might not only depend on the properties of the hormones themselves, as usually expected, but also on some features (e.g. species specificity, sensitivity) of the bioassays used for their functional characterization.Studies on lactogens are most often performed using the rat Nb2 cell proliferation bioassay (10Goffin V. Norman M. Martial J.A. Mol. Endocrinol. 1992; 6: 1381-1392PubMed Google Scholar, 11Goffin V. Struman I. Goormaghtigh E. Martial J.A. Eur. J. Biochem. 1993; 214: 483-490Crossref PubMed Scopus (15) Google Scholar, 12Goffin V. Struman I. Mainfroid V. Kinet S. Martial J.A. J. Biol. Chem. 1994; 269: 32598-32606Abstract Full Text PDF PubMed Google Scholar, 14Kinet S. Goffin V. Mainfroid V. Martial J.A. J. Biol. Chem. 1996; 271: 14353-14360Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 27Binder L. Gertler A. Elberg G. Guy R. Vogel T. Mol. Endocrinol. 1990; 4: 1060-1068Crossref PubMed Scopus (21) Google Scholar, 28Gout P.W. Beer C.T. Noble R.L. Cancer Res. 1980; 40: 2433-2436PubMed Google Scholar, 29Tanaka T. Shiu R.P.C. Gout P.W. Beer C.T. Noble R.L. Friesen H.G. J. Clin. Endocrinol. & Metab. 1980; 51: 1058-1063Crossref PubMed Scopus (565) Google Scholar, 30Luck D.N. Gout P.W. Kelsay K. Atkinson T. Beer C.T. Smith M. Mol. Endocrinol. 1990; 4: 1011-1016Crossref PubMed Scopus (18) Google Scholar, 31Luck D.N. Huyer M. Gout P.W. Beer C.T. Smith M. Mol. Endocrinol. 1991; 5: 1880-1886Crossref PubMed Scopus (27) Google Scholar). In order to investigate if the use of this bioassay might be misleading for assessing antagonistic properties of lactogen analogs, such as G129R hPRL (12Goffin V. Struman I. Mainfroid V. Kinet S. Martial J.A. J. Biol. Chem. 1994; 269: 32598-32606Abstract Full Text PDF PubMed Google Scholar), we have analyzed several PRLs, wild type (WT) or mutated, using an in vitro bioassay in which the transcriptional activation of PRL-responsive reporter genes is mediated by exogenous PRLR from rat or human species, which allows their comparison in a strictly identical cell system. Our data clearly show that (i) the rat PRLR displays a higher affinity than human PRLR for binding site 2 of PRLs, (ii) consequently, the agonistic and antagonistic properties of a given analog are driven by species specific features, and (iii) the hPRL analog G129R displays antagonistic properties on PRLR from both species, in to we previously observed using the Nb2 proliferation bioassay (12Goffin V. Struman I. Mainfroid V. Kinet S. Martial J.A. J. Biol. Chem. 1994; 269: 32598-32606Abstract Full Text PDF PubMed Google Scholar).