[en] For many years, our group has been involved in the development of human PRL antagonists. In two recent publications, S179D-human PRL, a human PRL analog designed to mimic a putative S179-phosphorylated human PRL, was reported to be a highly potent antagonist of human PRL-induced proliferation and signaling in rat Nb2 cells. We prepared this analog with the aim of testing it in various bioassays involving the homologous, human PRL receptor. In our hands, S179D- human PRL was able to stimulate 1) the proliferation of rat Nb2 cells and of human mammary tumor epithelial cells (T-47D), 2) transcriptional activation of the lactogenic hormone response element-luciferase reporter gene, and 3) activation of the Janus kinase/signal transducer and activator of transcription and MAPK pathways. Using the previously characterized antagonist G129R-human PRL as a control, we failed to observe any evidence for antagonism of S179D-human PRL toward any of the human PRL-induced effects analyzed, including cell proliferation, transcriptional activation, and signaling. In conclusion, our data argue that S179D-human PRL is an agonist displaying slightly reduced affinity and activity due to local alteration of receptor binding site 1, and that the antagonistic properties previously attributed to S179D-human PRL cannot be confirmed in any of the assays analyzed in this study.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Bernichtein, Sophie; INSERM, U-344
Kinet, Sandrina
Jeay, Sébastien; INSERM, U-344
Llovera, Marta; INSERM, U-344
Madern, Dominique
Martial, Joseph ; Université de Liège - ULiège > Département des sciences de la vie > GIGA-R : Biologie et génétique moléculaire
Kelly, Paul A.; INSERM, U-344
Goffin, Vincent; Centre National de la Recherche Scientifique - CNRS
Language :
English
Title :
S179D-human PRL, a pseudophosphorylated human PRL analog, is an agonist and not an antagonist
Publication date :
2001
Journal title :
Endocrinology
ISSN :
0013-7227
eISSN :
1945-7170
Publisher :
Endocrine Society, Chevy Chase, United States - Maryland
Bole-Feysot C., Goffin V., Edery M., Binart N., Kelly P.A. (1998) Prolactin and its receptor: Actions, signal transduction pathways and phenotypes observed in prolactin receptor knockout mice. Endocr Rev 19:225-268.
Horseman N.D., Zhao W., Montecino-Rodriguez E. (1997) Defective mammopoiesis, but normal hematopoiesis, in mice with a targeted disruption of the prolactin gene. EMBO J 16:6926-6935.
Ormandy C.J., Camus A., Barra J. (1997) Null mutation of the prolactin receptor gene produces multiple reproductive defects in the mouse. Genes Dev 11:167-178.
Kelly P.A., Djiane J., Postel-Vinay M.C., Edery M. (1991) The prolactin/growth hormone receptor family. Endocr Rev 12:235-251.
Goffin V., Kelly P.A. (1996) Prolactin and growth hormone receptors. Clin Endocrinol (Oxf) 45:247-255.
Goffin V., Shiverick K.T., Kelly P.A., Martial J.A. (1996) Sequence-function relationships within the expanding family of prolactin, growth hormone, placental lactogen and related proteins in mammals. Endocr Rev 17:385-410.
Wells J.A. (1996) Binding in the growth hormone receptor complex. Proc Natl Acad Sci USA 93:1-6.
Llovera M., Touraine P., Kelly P.A., Goffin V. (2000) Involvement of prolactin in breast cancer: Redefining the molecular targets. Exp Gerontol 35:41-51.
Lebrun J.J., Ali S., Sofer L., Ullrich A., Kelly P.A. (1994) Prolactin-induced proliferation of Nb2 cells involves tyrosine phosphorylation of the prolactin receptor and its associated tyrosine kinase JAK2. J Biol Chem 269:14021-14026.
Rui H., Lebrun J.J., Kirken R.A., Kelly P.A., Farrar W.L. (1994) Jak2 activation and cell proliferation induced by antibody-mediated prolactin receptor dimerization. Endocrinology 135:1299-1306.
Wakao H., Gouilleux F., Groner B. (1994) Mammary gland factor (MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response. EMBO J 13:2182-2191.
Sinha Y.N. (1995) Structural variants of prolactin: Occurence and physiological significance. Endocr Rev 16:354-369.
Brooks C.L., Kim B.G., Aphale P., Kleeman B.E., Johnson G.C. (1990) Phosphorylated variant of bovine prolactin. Mol Cell Endocrinol 71:117-123.
Kim B.G., Brooks C.L. (1993) Isolation and characterization of phosphorylated bovine prolactin. Biochem J 296:41-47.
Mastro R.M., Dannies P.S. (1995) Lack of correlation of distribution of prolactin (PRL) charge isoforms with induction of PRL storage. Endocrinology 136:69-74.
Wang Y.F., Liu J.W., Mamidi M., Walker A.M. (1996) Identification of the major site of rat prolactin phosphorylation as serine 177. J Biol Chem 271:2462-2469.
Wang Y.F., Walker A.M. (1993) Dephosphorylation of standard prolactin produces a more biologically active molecule: Evidence for antagonism between nonphosphorylated and phosphorylated prolactin in the stimulation of Nb2 cell proliferation. Endocrinology 133:2156-2160.
Chen T.J., Kuo C.B., Tsai K.F., Liu J.W., Chen D.Y., Walker A.M. (1998) Development of recombinant human prolactin receptor antagonists by molecular mimicry of the phosphorylated hormone. Endocrinology 139:609-616.
Wicks J.R., Brooks C.L. (1995) Biological activity of phosphorylated and dephosphorylated bovine prolactin. Mol Cell Endocrinol 112:223-229.
Luck D.N., Gout P.W., Beer C.T., Smith M. (1989) Bioactive recombinant methionyl bovine prolactin: Structure-function studies using site-specific mutagenesis. Mol Endocrinol 3:822-831.
Fuh G., Cunningham B.C., Fukunaga R., Nagata S., Goeddel D.V., Wells J.A. (1992) Rational design of potent antagonists to the human growth hormone receptor. Science 256:1677-1680.
Fuh G., Colosi P., Wood W.I., Wells J.A. (1993) Mechanism-based design of prolactin receptor antagonists. J Biol Chem 268:5376-5381.
Goffin V., Kinet S., Ferrag F., Binart N., Martial J.A., Kelly P.A. (1996) Antagonistic properties of human prolactin analogs that show paradoxical agonistic activity in the Nb2 bioassay. J Biol Chem 271:16573-16579.
Kinet S., Bernichtein S., Kelly P.A., Martial J.A., Goffin V. (1999) Biological properties of human prolactin analogs depend not only on global hormone affinity, but also on the relative affinities of both receptor binding sites. J Biol Chem 274:26033-26043.
Llovera M., Pichard C., Bernichtein S. (2000) Human prolactin (hPRL) antagonists inhibit hPRL-activated signaling pathways involved in breast cancer cell proliferation. Oncogene 19:4695-4705.
Coss D., Kuo C.B., Yang L., Ingleton P., Luben R., Walker A.M. (1999) Dissociation of Janus kinase 2 and signal transducer and activator of transcription 5 activation after treatment of Nb2 cells with a molecular mimic of phosphorylated prolactin. Endocrinology 140:5087-5094.
Goffin V., Touraine P., Pichard C., Bernichtein S., Kelly P.A. (1999) Should prolactin be reconsidered as a therapeutic target in human breast cancer?. Mol Cell Endocrinol 151:79-87.
Cooke N.E., Coit D., Shine J., Baxter J.D., Martial J.A. (1981) Human prolactin: cDNA structural analysis and evolutionary comparisons. J Biol Chem 256:4007-4016.
Goffin V., Struman I., Mainfroid V., Kinet S., Martial J.A. (1994) Evidence for a second receptor binding site on human prolactin. J Biol Chem 269:32598-32606.
Paris N., Rentier-DeIrue F., Defontaine A. (1990) Bacterial production and purification of recombinant human prolactin. Biotechnol Appl Biochem 12:436-449.
Goffin V., Norman M., Martial J.A. (1992) Alanine-scanning mutagenesis of human prolactin: Importance of the 58-74 region for bioactivity. Mol Endocrinol 6:1381-1392.
Lochnan H.A., Buteau H., Richards S., Edery M., Kelly P.A. (1995) Functional activity of the human prolactin receptor and its ligands. Mol Cell Endocrinol 114:91-99.
Laemmli U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.
Andrade M.A., Chacon P., Merelo J.J., Moran F. (1993) Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Engin 6:383-390.
Tanaka T., Shiu R.P., Gout P.W., Beer C.T., Noble R.L., Friesen H.G. (1980) A new sensitive and specific bioassay for lactogenic hormones: Measurement of prolactin and growth hormone in human serum. J Clin Endocrinol Metab 51:1058-1063.
Gout P.W., Beer C.T., Noble R.L. (1980) Prolactin-stimulated growth of cell cultures established from malignant Nb rat lymphomas. Cancer Res 40:2433-2436.
Goffin V., Bernichtein S., Carrière O., Bennett W.F., Kopchick J.J., Kelly P.A. (1999) The human growth hormone antagonist B2036 does not interact with the prolactin receptor. Endocrinology 140:3853-3856.
Goffin V., Struman I., Goormaghtigh E., Martial J.A. (1993) The addition of nine residues at the C-terminus of human prolactin drastically alters its biological properties. Eur J Biochem 214:483-490.
Kinet S., Goffin V., Mainfroid V., Martial J.A. (1996) Characterization of lactogen receptor binding site 1 of human prolactin. J Biol Chem 271:14353-14360.
Shiu R.P., Elsholtz H.P., Tanaka T. (1983) Receptor-mediated mitogenic action of prolactin in a rat lymphoma cell line. Endocrinology 113:159-165.
Dattani M.T., Hindmarsh P.C., Brook C.D., Robinson I.F., Kopchick J.J., Marshall N.J. (1995) G120R, a human growth hormone antagonist, shows zinc-dependent agonist and antagonist activity on Nb2 cells. J Biol Chem 270:9222-9226.
Gertler A., Shamay A., Cohen N. (1986) Inhibition of lactogenic activities of ovine prolactin and human growth hormone (hGH) by a novel form of modified recombinant hGH. Endocrinology 118:720-726.
Binder L., Gertler A., Elberg G., Guy R., Vogel T. (1990) Site-directed mutations of human growth hormone that selectively modify its lactogenic activity and binding properties. Mol Endocrinol 4:1060-1068.
Sakal E., Elberg G., Gertler A. (1997) Direct evidence that lactogenic hormones induce homodimerization of membrane-anchored prolactin receptor in intact Nb2-11C rat lymphoma cells. FEBS Lett 410:289-292.
Fuh G., Wells J.A. (1995) Prolactin receptor antagonists that inhibit the growth of breast cancer cell lines. J Biol Chem 270:13133-13137.
Shiu R.C., Elsholtz H.P., Tanaka T. (1983) Receptor-mediated mitogenic action of prolactin in a rat lymphoma cell line. Endocrinology 113:159-165.
Ilondo M.M., Damholt A.B., Cunningham B.A., Wells J.A., De Meyts P., Shymko R.M. (1994) Receptor dimerization determines the effects of growth hormone in primary rat adipocytes and cultured human IM-9 lymphocytes. Endocrinology 134:2397-2403.
Gouilleux F., Wakao H., Mundt M., Groner B. (1994) Prolactin induces phosphorylation of Tyr694 of Stat5 (MGF), a prerequisite for DNA binding and induction of transcription. EMBO J 13:4361-4369.
Goffin V., Martial J.A., Summers N.L. (1995) Use of a model to understand prolactin and growth hormone specificities. Prot Engin 8:1215-1231.
Yang L., Kuo C.B., Liu Y. (2001) Administration of unmodified prolactin (U-PRL) and a molecular mimic of phosphorylated prolactin (PP-PRL) during rat pregnancy provides evidence that the U-PRL:PP-PRL ratio is crucial to the normal development of pup tissues. J Endocrinol 168:227-238.
Xu X.L., Kreye E., Kuo C.B., Yang L., Walker A.M. Administration of human PRL mutant, S179D, markedly reduces the incidence of tumors when DU145 human prostate cancer cells are grown in nude mice. Proceedings of the 82nd Annual Meeting of The Endocrine Society , Toronto, Canada; 2000.
Bridges R.S., Rigero B.A., Byrnes E.M., Yang L., Walker A.M. (2001) Central infusions of the recombinant human prolactin receptor antagonist, S179D-PRL, delay the onset of maternal behavior in steroid- primed, nulliparous female rats. Endocrinology 142:730-739.
Yang L., Kuo C.B., Xu X.L., Coss D., Walker A.M. Administration of a prolactin receptor antagonist to pregnant rats resulted in increased fetal intrathymic apoptosis and no γδ T cell seeding of the epidermis. Proceedings of the 82nd Annual Meeting of The Endocrine Society , Toronto, Canada; 2000.
Chen C., Xu X.L., Yang L., Coss D., Walker A.M. Unmodified prolactin promotes ductal and lobulo-alveolar growth, while a molecular mimic of phosphorylated prolactin promotes lobuloalveolus formation and casein gene expression in the pregnant rat mammary gland. Proceedings of the 82nd Annual Meeting of The Endocrine Society , Toronto, Canada; 2000.
Coss D., Yang L., Kuo C.B., Xu X., Luben R.A., Walker A.M. (2000) Effects of prolactin on osteoblast alkaline phosphatase and bone formation in the developing rat. Am J Physiol 279:1216-1225.
Clement-Lacroix P., Ormandy C., Lepescheux L. (1999) Osteoblasts are a new target for prolactin: Analysis of bone formation in prolactin receptor knockout mice. Endocrinology 140:96-105.
