[en] We report the solution structure of human prolactin determined by NMR spectroscopy. Our result is a significant improvement over a previous structure in terms of number and distribution of distance restraints, regularity of secondary structure, and potential energy. More significantly, the structure is sufficiently different that it leads to different conclusions regarding the mechanism of receptor activation and initiation of signal transduction. Here, we compare the structure of unbound prolactin to structures of both the homologue ovine placental lactogen and growth hormone. The structures of unbound and receptor bound prolactin/placental lactogen are similar and no noteworthy structural changes occur upon receptor binding. The observation of enhanced binding at the second receptor site when the first site is occupied has been widely interpreted to indicate conformational change induced by binding the first receptor. However, our results indicate that this enhanced binding at the second site could be due to receptor-receptor interactions or some other free energy sources rather than conformational change in the hormone. Titration of human prolactin with the extracellular domain of the human prolactin receptor was followed by NMR, gel filtration and electrophoresis. Both binary and ternary hormone-receptor complexes are clearly detectable by gel filtration and electrophoresis. The binary complex is not observable by NMR, possibly due to a dynamic equilibrium in intermediate exchange within the complex. The ternary complex of one hormone molecule bound to two receptor molecules is on the contrary readily detectable by NMR. This is in stark contrast to the widely held view that the ternary prolactin-receptor complex is only transiently formed. Thus, our results lead to improved understanding of the prolactin-prolactin receptor interaction.
Research Center/Unit :
Giga-Development and Stem Cells - ULiège
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Teilum, K.; Institute of Molecular Biology and Physiology, University of Copenhagen > Department of Protein Chemistry
Hoch, J. C.; University of Connecticut Health Center Farmington > Department of Molecular Microbial and Structural Biology
Goffin, Vincent; Université Paris Descartes / Université de Liège
Kinet, Sandrina; Université de Liège - ULiège
Martial, Joseph ; Université de Liège - ULiège > Département des sciences de la vie > GIGA-R : Biologie et génétique moléculaire
Kragelund, B. B.; Institute of Molecular Biology and Physiology University of Copenhagen > Department of Protein Chemistry
C. Bole-Feysot, V. Goffin, M. Edery, N. Binart, and P.A. Kelly Prolactin (PRL) and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice Endocr. Rev. 19 1998 225 268
U. Sivaprasad, J.M. Canfield, and C.L. Brooks Mechanism for ordered receptor binding by human prolactin Biochemistry 43 2004 13755 13765
A. Gertler, and J. Djiane Mechanism of ruminant placental lactogen action: molecular and in vivo studies Mol. Genet. Metab. 75 2002 189 201
B.C. Cunningham, and J.A. Wells High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis Science 244 1989 1081 1085
V. Goffin, M. Norman, and J.A. Martial Alanine-scanning mutagenesis of human prolactin: importance of the 58-74 region for bioactivity Mol. Endocrinol. 6 1992 1381 1392
S. Kinet, V. Goffin, V. Mainfroid, and J.A. Martial Characterization of lactogen receptor-binding site 1 of human prolactin J. Biol. Chem. 271 1996 14353 14360
W. Somers, M. Ultsch, A.M. de Vos, and A.A. Kossiakoff The X-ray structure of a growth hormone-prolactin receptor complex Nature 372 1994 478 481
B.C. Cunningham, and J.A. Wells Rational design of receptor-specific variants of human growth hormone Proc. Natl Acad. Sci. USA 88 1991 3407 3411
D.N. Luck, M. Huyer, P.W. Gout, C.T. Beer, and M. Smith Single amino acid substitutions in recombinant bovine prolactin that markedly reduce its mitogenic activity in Nb2 cell cultures Mol. Endocrinol. 5 1991 1880 1886
S. Bernichtein, C. Kayser, K. Dillner, S. Moulin, J.J. Kopchick, and J.A. Martial Development of pure prolactin receptor antagonists J. Biol. Chem. 278 2003 35988 35999
V. Goffin, S. Bernichtein, C. Kayser, and P.A. Kelly Development of new prolactin analogs acting as pure prolactin receptor antagonists Pituitary 6 2003 89 95
S. Bernichtein, S. Kinet, S. Jeay, M. Llovera, D. Madern, and J.A. Martial S179D-human PRL, a pseudophosphorylated human PRL analog, is an agonist and not an antagonist Endocrinology 142 2001 3950 3963
N. Ferrara, C. Clapp, and R. Weiner The 16K fragment of prolactin specifically inhibits basal or fibroblast growth factor stimulated growth of capillary endothelial cells Endocrinology 129 1991 896 900
C. Clapp, J.A. Martial, R.C. Guzman, F. Rentier-Delrue, and R.I. Weiner The 16-kilodalton N-terminal fragment of human prolactin is a potent inhibitor of angiogenesis Endocrinology 133 1993 1292 1299
F. Bentzien, I. Struman, J.F. Martini, J. Martial, and R. Weiner Expression of the antiangiogenic factor 16K hPRL in human HCT116 colon cancer cells inhibits tumor growth in Rag1(-/-) mice Cancer Res. 61 2001 7356 7362
M.A. Rycyzyn, and C.V. Clevenger The intranuclear prolactin/cyclophilin B complex as a transcriptional inducer Proc. Natl Acad. Sci. USA 99 2002 6790 6795
M.A. Rycyzyn, S.C. Reilly, K. O'Malley, and C.V. Clevenger Role of cyclophilin B in prolactin signal transduction and nuclear retrotranslocation Mol. Endocrinol. 14 2000 1175 1186
P. Carter, C.A. Andersen, and B. Rost DSSPcont: continuous secondary structure assignments for proteins Nucl. Acids Res. 31 2003 3293 3295
A. Gertler, J. Grosclaude, C.J. Strasburger, S. Nir, and J. Djiane Real-time kinetic measurements of the interactions between lactogenic hormones and prolactin-receptor extracellular domains from several species support the model of hormone-induced transient receptor dimerization J. Biol. Chem. 271 1996 24482 24491
C. Keeler, P.S. Dannies, and M.E. Hodsdon The tertiary structure and backbone dynamics of human prolactin J. Mol. Biol. 328 2003 1105 1121
M.J. Sippl Recognition of errors in three-dimensional structures of proteins Proteins: Struct. Funct. Genet. 17 1993 355 362
R.W. Hooft, G. Vriend, C. Sander, and E.E. Abola Errors in protein structures Nature 381 1996 272
W.D. Cornell, P. Cieplak, C.I. Bayly, I.R. Gould, K.M. Merz, and D.M. Ferguson A 2Nd generation force-field for the simulation of proteins. Nucleic-acids, and organic-molecules J. Am. Chem. Soc. 117 1995 5179 5197
W.D. Cornell, P. Cieplak, C.I. Bayly, I.R. Gould, K.M. Merz, and D.M. Ferguson A second generation force field for the simulation of proteins, nucleic acids, and organic molecules (vol 117, pg 5179, 1995) J. Am. Chem. Soc. 118 1996 2309
P.A. Elkins, H.W. Christinger, Y. Sandowski, E. Sakal, A. Gertler, A.M. de Vos, and A.A. Kossiakoff Ternary complex between placental lactogen and the extracellular domain of the prolactin receptor Nature Struct. Biol. 7 2000 808 815
M.H. Ultsch, W. Somers, A.A. Kossiakoff, and A.M. de Vos The crystal structure of affinity-matured human growth hormone at 2 Å resolution J. Mol. Biol. 236 1994 286 299
T. Clackson, M.H. Ultsch, J.A. Wells, and A.M. de Vos Structural and functional analysis of the 1:1 growth hormone:receptor complex reveals the molecular basis for receptor affinity J. Mol. Biol. 277 1998 1111 1128
A.M. de Vos, M. Ultsch, and A.A. Kossiakoff Human growth hormone and extracellular domain of its receptor: crystal structure of the complex Science 255 1992 306 312
V. Goffin, I. Struman, V. Mainfroid, S. Kinet, and J.A. Martial Evidence for a second receptor binding site on human prolactin J. Biol. Chem. 269 1994 32598 32606
B.C. Cunningham, D.J. Henner, and J.A. Wells Engineering human prolactin to bind to the human growth hormone receptor Science 247 1990 1461 1465
P.L. Rouzic, O. Sandra, J. Grosclaude, F. Rentier-Delrue, O. Jolois, and M. Tujague Evidence of rainbow trout prolactin interaction with its receptor through unstable homodimerisation Mol. Cell Endocrinol. 172 2001 105 113
A. Tchelet, N.R. Staten, D.P. Creely, G.G. Krivi, and A. Gertler Extracellular domain of prolactin receptor from bovine mammary gland: expression in Escherichia coli, purification and characterization of its interaction with lactogenic hormones J. Endocrinol. 144 1995 393 403
E. Biener, C. Martin, N. Daniel, S.J. Frank, V.E. Centonze, and B. Herman Ovine placental lactogen-induced heterodimerization of ovine growth hormone and prolactin receptors in living cells is demonstrated by fluorescence resonance energy transfer microscopy and leads to prolonged phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT3 Endocrinology 144 2003 3532 3540
Y. Sandowski, M. Nagano, C. Bignon, J. Djiane, P.A. Kelly, and A. Gertler Preparation and characterization of recombinant prolactin receptor extracellular domain from rat Mol. Cell Endocrinol. 115 1995 1 11
T. Clackson, and J.A. Wells A hot spot of binding energy in a hormone-receptor interface Science 267 1995 383 386
D.N. Luck, P.W. Gout, K. Kelsay, T. Atkinson, C.T. Beer, and M. Smith Recombinant methionyl bovine prolactin: loss of bioactivity after single amino acid deletions from putative helical regions Mol. Endocrinol. 4 1990 1011 1016
Z. Sun, P.S. Li, P.S. Dannies, and J.C. Lee Properties of human prolactin (PRL) and H27A-PRL, a mutant that does not bind Zn2+ Mol. Endocrinol. 10 1996 265 271
V. Goffin, S. Kinet, F. Ferrag, N. Binart, J.A. Martial, and P.A. Kelly Antagonistic properties of human prolactin analogs that show paradoxical agonistic activity in the Nb2 bioassay J. Biol. Chem. 271 1996 16573 16579
V. Goffin, J.A. Martial, and N.L. Summers Use of a model to understand prolactin and growth hormone specificities Protein Eng. 8 1995 1215 1231
V. Goffin, K.T. Shiverick, P.A. Kelly, and J.A. Martial Sequence-function relationships within the expanding family of prolactin, growth hormone, placental lactogen, and related proteins in mammals Endocr. Rev. 17 1996 385 410
B. Bernat, G. Pal, M. Sun, and A.A. Kossiakoff Determination of the energetics governing the regulatory step in growth hormone-induced receptor homodimerization Proc. Natl Acad. Sci. USA 100 2003 952 957
S.T. Walsh, L.M. Jevitts, J.E. Sylvester, and A.A. Kossiakoff Site2 binding energetics of the regulatory step of growth hormone-induced receptor homodimerization Protein Sci. 12 2003 1960 1970
S. Bernichtein, J.B. Jomain, P.A. Kelly, and V. Goffin The N-terminus of human prolactin modulates its biological properties Mol. Cell Endocrinol. 208 2003 11 21
A. Morin, R. Picart, and A. Tixier-Vidal Effects of the N-terminal cysteine mutation on prolactin expression and secretion in transfected cells Mol. Cell Endocrinol. 117 1996 59 73
F.C. Peterson, and C.L. Brooks Identification of a motif associated with the lactogenic actions of human growth hormone J. Biol. Chem. 272 1997 21444 21448
F.C. Peterson, and C.L. Brooks Different elements of mini-helix 1 are required for human growth hormone or prolactin action via the prolactin receptor Protein Eng. Des. Sel. 17 2004 417 424
K.M. Duda, and C.L. Brooks Differential effects of zinc on functionally distinct human growth hormone mutations Protein Eng 16 2003 531 534
K.M. Duda, and C.L. Brooks Human growth hormone site 2 lactogenic activity requires a distant tyrosine164 FEBS Letters 449 1999 120 124
M.R. Kasimova, S.M. Kristensen, P.W. Howe, T. Christensen, F. Matthiesen, and J. Petersen NMR studies of the backbone flexibility and structure of human growth hormone: a comparison of high and low pH conformations J. Mol. Biol. 318 2002 679 695
N. Paris, F. Rentier-Delrue, A. Defontaine, V. Goffin, J.J. Lebrun, L. Mercier, and J.A. Martial Bacterial production and purification of recombinant human prolactin Biotechnol. Appl. Biochem. 12 1990 436 449
M.H. Lerche, A. Meissner, F.M. Poulsen, and O.W. Sorensen Pulse sequences for measurement of one-bond N-15-H-1 coupling constants in the protein backbone J. Magn. Reson. 140 1999 259 263
F. Delaglio, S. Grzesiek, G.W. Vuister, G. Zhu, J. Pfeifer, and A. Bax NMRPipe: a multidimensional spectral processing system based on UNIX pipes J. Biomol. NMR 6 1995 277 293
M. Kjær, K.V. Andersen, and F.M. Poulsen Automated and semiautomated analysis of homo- and heteronuclear multidimensional nuclear magnetic resonance spectra of proteins: the program Pronto Methods Enzymol. 239 1994 288 307
T. Herrmann, P. Guntert, and K. Wuthrich Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA J. Mol. Biol. 319 2002 209 227
C.D. Schwieters, J.J. Kuszewski, N. Tjandra, and G.M. Clore The Xplor-NIH NMR molecular structure determination package J. Magn. Reson. 160 2003 65 73
J. Kuszewski, A.M. Gronenborn, and G.M. Clore Improvements and extensions in the conformational database potential for the refinement of NMR and X-ray structures of proteins and nucleic acids J. Magn. Reson. 125 1997 171 177
R. Koradi, M. Billeter, and K. Wuthrich MOLMOL: a program for display and analysis of macromolecular structures J. Mol. Graph. 14 1996 51 55
A. Sali, and T.L. Blundell Definition of general topological equivalence in protein structures. A procedure involving comparison of properties and relationships through simulated annealing and dynamic programming J. Mol. Biol. 212 1990 403 428