Prolactin (PRL) interacts with a specific, well characterized plasma membrane receptor (PRLR) that is coupled to signal transduction pathways involving Jak2, Fyn, and MAP kinases, and signal transducers and activators of transcription (STAT). Although a few previous studies have indicated nuclear translocation of PRL in IL-2 stimulated T lymphocytes, PRL-dependent Nb2 lymphoma cell lines and 235–1 lactotrophs, the mechanisms of nuclear targeting remain unknown and conflicting results have been reported concerning the putative nuclear translocation of the PRLR. We therefore decided to investigate nuclear translocation of PRLR and PRL in various cell lines transfected with an expression plasmid encoding PRLR, using confocal laser microscopy. We have constructed various cDNAs of the long and short forms of the rat PRLR containing an oligonucleotide encoding a Flag epitope inserted either just before the N-terminal amino acid or in the C-terminal end of the mature receptor (named N-terminal or C-terminal Flag-tagged PRLR). The corresponding receptors function as the PRLR in transfected cells: they are expressed at the plasma membrane and in compartments of the secretory pathway, they bind PRL with normal affinity (Kd= 4x10(−10) M) and have the same capacity to stimulate the transcriptional activity of a milk protein (beta-casein) gene as wild-type PRLR. In addition, the tagged receptors are much more efficiently immunodetected using anti-Flag antibodies, as compared to anti-PRL antibodies (U5 or U6). Immunofluorescence combined with detailed confocal laser microscopy showed that addition of PRL (0 to 12 hours) to COS-7, CHO and NIH-3T3 transfected fibroblasts induces rapid internalization of the receptor (long form), without any translocation to the nucleus. Using PRL-R tagged both in the N-terminal or C-terminal regions of the mature receptor excludes the possibility of a cleaved fragment which could have been subsequently imported into the nucleus. An absence of nuclear translocation of PRLR was also observed in a 293 cell line stably expressing the receptor, and in physiological targets for PRL, i.e. in Nb2 lymphoma cells expressing the Nb2 form of the receptor or in BGME mammary gland epithelial cells upon overexpression of a Flag-tagged PRLR. Similarly, the short form of the PRLR was not detected in nuclei of transfected COS cells upon PRL treatment. Clearly, our results provide evidence that internalization of the plasma membrane PRLR does not lead to nuclear translocation of the receptor, or part of it, in most fibroblasts and epithelial cells at physiological concentrations of PRL. Also, in co-localization experiments, PRL was internalized without nuclear translocation. Activation of STATs transcription factors and MAP kinases, as well as translocation of these proteins to the nucleus following their phosphorylation, probably remains the intracellular mechanism coupling stimulation to nuclear events.

Bader
T.
,
Wietzerbin
J.
(
1994
).
Nuclear accumulation of interferon.
Proc. Nat. Acad. Sci. USA
91
,
11831
11835
Baldin
V.
,
Roman
A. M.
,
Basc-Bieme
I.
,
Amalvic
F.
,
Bouche
G.
(
1990
).
Translocation of bFGF to the nucleus in G1 phase cell cycle specific in aortic endothelial cells.
EMBO J
9
,
1511
1518
Bazan
F.
(
1989
).
A novel family of growth factor receptors: a common binding domain in the growth hormone, prolactin, erythropoietin and IL-6 receptors, and p75 IL-2 receptorchain.
Biochem. Biophys. Res. Commun
164
,
788
795
Bergeron
J. J. M.
,
Resch
L.
,
Rachubinski
R.
,
Patel
B. A.
,
Poster
B. I.
(
1983
).
Effect of colchicine on internalization of prolactin in female rat liver: an in vivo radioautographic study.
J. Cell Biol
96
,
875
886
Berlanga
J. J.
,
Gualillo
O.
,
Buteau
H.
,
Applanat
M.
,
Kelly
P.
,
Edery
M.
(
1997
).
Prolactin activates tyrosyl phosphorylation of insulin receptor substrate 1 and phosphatidylinositol-3-OH kinase.
J. Biol. Chem
272
,
2050
2052
Bouche
G.
,
Gas
N.
,
Prats
H.
,
Baldin
V.
,
Tauber
J. P.
,
Teissie
J.
,
Almaric
F.
(
1987
).
Basic fibroblast growth factor enters the nucleolus and stimulates transcription of ribosomal genes in ABAE cells undergoing G0 to G1 transition.
Proc. Nat. Acad. Sci. USA
84
,
6770
6774
Buckley
A. R.
,
Crowe
P. D.
,
Russel
D. H.
(
1988
).
Rapid activation of protein kinase C in isolated rat liver nuclei by prolactin, a known hepatic mitogen.
Proc. Nat. Acad. Sci USA
85
,
8649
8653
Burwen
S. J.
,
Jones
A. L.
(
1987
).
The association of polypeptide hormones and growth factors with the nuclei of target cells.
Trends Biochem. Sci
12
,
59
162
Clevenger
C. V.
,
Russel
D. H.
,
Appasamy
P. M.
,
Prystowsky
B.
(
1990
).
Regulation of interleukin 2-driven T-lymphocytes proliferation by prolactin.
Proc. Nat. Acad. Sci. USA
87
,
6460
6464
Clevenger
C. V.
,
Sillman
A. L.
,
Prystowsky
B.
(
1990
).
Interleukin-2 driven nuclear translocation of prolactin in cloned T-lymphocytes.
Endocrinology
127
,
3151
3159
Clevenger
C. V.
,
Altmann
S. W.
,
Prystowsky
M. B.
(
1991
).
Requirement of nuclear prolactin for interleukin-2 stimulated proliferation of T-lymphocytes.
Science
253
,
77
79
Curtis
B. M.
,
Widmer
M. B.
,
De Roos
P.
,
Quarnstrom
E. E.
(
1990
).
IL-1 and its receptor are translocated to the nucleus.
J. Immunol
144
,
1295
1303
Darnell
J. E.
,
Kerr
I. M.
,
Stark
G. R.
(
1994
).
Jak STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins.
Science
264
,
1415
1421
Das
R.
,
Vonderhaar
B. K.
(
1995
).
Transduction of prolactin's signal through both long and short forms of the prolactin receptor.
Mol. Endocrinol
9
,
1750
1759
Davis
J. A.
,
Linzer
D. I. H.
(
1988
).
Autocrine stimulation of Nb2 cells proliferation by secreted, but not intracellular, prolactin.
Mol. Endocrinol
2
,
740
746
Dingwall
C.
,
Laskey
R. A.
(
1991
).
Nuclear targeting sequence- a consensus?.
Trends Biochem. Sci
16
,
478
481
Djiane
J.
,
Delouis
D.
,
Kelly
P. A.
(
1982
).
Prolactin receptors in explants of pseudopregnant rabbit mammary glands.
Mol. Cell. Endocrinol
25
,
163
170
Dusanter-Fourt
I.
,
Muller
O.
,
Ziemiecki
A.
,
Mayeux
P.
,
Drucker
B.
,
Djiane
J.
,
Wilks
A.
,
Harpur
A. G.
,
Fisher
S.
,
Gisselbrecht
S.
(
1994
).
Identification of JAK protein tyrosine kinases as signaling molecules for prolactin. Functional analysis of prolactin receptor and prolactin-erythropoietin receptor chimera expressed in lymphoid cells.
EMBOJ
13
,
2583
2591
Ellis
L.
,
Clauser
E.
,
Morgan
D. O.
,
Edery
M.
,
Roth
R. A.
,
Rutter
W. J.
(
1986
).
Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose.
Cell
45
,
721
732
Ginsburg
E.
,
Vonderhaar
B. K.
(
1995
).
Prolactin synthesis and secretion by human breast cancer cells.
Cancer Res
55
,
2591
2595
Goldfine
I. D.
,
Smith
G. J.
,
Wong
K. Y.
,
Jones
A. L.
(
1977
).
Cellular uptake and nuclear binding of insulin in human cultured lymphocytes: evidence for potential intracellular sites of insulin action.
Proc. Nat. Acad. Sci USA
74
,
1368
1372
Goujon
L.
,
Allevato
G.
,
Simonin
G.
,
Paquereau
L.
,
Le Cam
A.
,
Clark
J.
,
Nielsen
J. H.
,
Djiane
J.
,
Postel-Vinay
M. C.
,
Edery
M.
,
Kelly
P. A.
(
1994
).
Cytoplasmic sequences of the growth hormone receptor necessary for signal transduction.
Proc. Nat. Acad. Sci. USA
91
,
957
961
Grenfell
S.
,
Smithers
N.
,
Miller
K.
,
Solari
R.
(
1989
).
Receptor-mediated endocytosis and nuclear transport of human interleukin 1-.
Biochem. J
264
,
813
822
James
R.
,
Bradshaw
R. A.
(
1984
).
Polypeptide growth factors.
Annu. Rev. Biochem
53
,
259
292
Johnson
L. K.
,
Vlodavsky
I.
,
Baxter
J. D.
,
Gospodarowicz
D.
(
1980
).
Nuclear accumulation of epidermal growth factor in cultured rat pituitary cells.
Nature
287
,
343
348
Josefsberg
Z.
,
Posner
B. I.
,
Patel
B.
,
Bergeron
J. J. M.
(
1979
).
The uptake of prolactin into female rat liver: concentration of intact hormone in the Golgi apparatus.
J. Biol. Chem
254
,
209
214
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
Lebrun
J. J.
,
Ali
S.
,
Ullrich
A.
,
Kelly
P. A.
(
1994
).
Proline-rich sequence-mediated Jak2 association to the prolactin receptor is required but not sufficient for signal transduction.
J. Biol. Chem
270
,
10664
10670
Lesueur
L.
,
Edery
M.
,
Ali
S.
,
Paly
J.
,
Kelly
P. A.
,
Djiane
J.
(
1991
).
Comparison of long and short forms of the prolactin receptor on prolactin-induced milk protein gene transcription.
Proc. Nat. Acad. Sci. USA
88
,
824
828
Lobie
P.
,
Barnard
R.
,
Waters
M. J.
(
1991
).
The nuclear growth hormone receptor-binding protein. Antigenic and physicochemical characterization.
J. Biol. Chem
266
,
1
5
Lobie
P.
,
Mertani
H.
,
Morel
G.
,
Morales-Bustos
O.
,
Norsteedt
G.
,
Waters
M. J.
(
1994
).
Receptor mediated nuclear translocation of growth hormone.
J. Biol. Chem
269
,
21330
21339
Lobie
P.
,
Wood
T. J.
,
Min Chen
C.
,
Waters
M. J.
,
Norsteedt
G.
(
1994
).
Nuclear translocation and anchorage of the growth hormone receptor.
J. Biol. Chem
269
,
31735
31746
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
Maher
D. W.
,
Lee
B. A.
,
Donoghue
D. J.
(
1989
).
The alternatively spliced exon of the platelet-derived growth factor A chain encodes a nuclear targeting signal.
Mol. Cell. Biol
9
,
2251
2253
Miller
D. S.
(
1988
).
Stimulation of RNA and protein synthesis of intracellular insulin.
Science
240
,
506
509
Morel
G.
(
1994
).
Internalization and nuclear localization of peptide hormone.
Biochem. Pharmacol
47
,
63
76
Murakami
M.
,
Narazaki
M.
,
Hibi
M.
,
Yasukawa
K.
,
Hamaguchi
M.
,
Taga
T.
,
Kishimoto
T.
(
1991
).
Critical cytoplasmic region of the interleukin 6 signal transducer gp 130 is conserved in the cytokine receptor family.
Proc. Nat. Acad. Sci. USA
88
,
11349
11353
Okamura
H.
,
Zachwieja
J.
,
Raguet
S.
,
Kelly
P. A.
(
1989
).
Characterization and application of monoclonal antibodies to the PRL receptor.
Endocrinology
124
,
2499
2508
Peralta Soler
A.
,
Thompson
K. A.
,
Smith
R. M.
,
Jarett
L.
(
1989
).
Immunological demonstration of the accumulation of insulin, but not insulin receptors, in nuclei of insulin-treated cells.
Proc. Nat. Acad. Sci. USA
86
,
6640
6644
Perrot-Applanat
M.
,
Lescop
P.
,
Milgrom
E.
(
1992
).
The cytoskeleton and the cellular traffic of the progesterone receptor.
J. Cell Biol
119
,
337
348
Perrot-Applanat
M.
,
Cibert
C.
,
Geraud
G.
,
Renoir
J.-M.
,
Baulieu
E. E.
(
1995
).
The 59 kDA FK506-binding protein, a 90 kDa heat shock protein binding immunophilin (FKBP59-HBI), is associated with the nucleus, the cytoskeleton and mitotic apparatus.
J. Cell Sci
108
,
2037
2051
Posner
B. I.
,
Bergeron
J. J. M.
,
Josefsberg
Z.
,
Khan
M. N.
,
Khan
R. J.
,
Patel
B. A.
,
Sikstrom
R. A.
,
Verma
A. K.
(
1981
).
Intracellular receptors and internalization.
Rec. Prog. Horm. Res
37
,
539
579
Rakowicz-Szulczynska
E. M.
,
Rodeck
U.
,
Herlyn
M.
,
Koprowski
H.
(
1986
).
Chromatin binding of epidermal growth factor, nerve growth factor, and platelet-derived growth factor in cells bearing the appropriate surface receptors.
Proc. Nat. Acad. Sci. USA
83
,
3723
3732
Rakowicz-Szulczynska
E. M.
(
1993
).
Identification of the cell surface and nuclear receptors for NGF in a breast carcinoma cell line 1.
J. Cell. Physiol
54
,
64
70
Rao
Y.
,
Olsonn
M. D.
,
Buckley
D. J.
,
Buckley
A. R.
(
1993
).
Nuclear co-localization of prolactin and the prolactin receptor in rat Nb2 node lymphoma cells.
Endocrinology
133
,
3062
3065
Sanger
F.
,
Nicklen
S.
,
Coulson
A. R.
(
1977
).
DNA sequencing with chain terminating inhibitors.
Proc. Nat. Acad. Sci USA
74
,
5463
5467
Savion
N.
,
Vlodavsky
I.
,
Gospodarowicz
D.
(
1981
).
Nuclear accumulation of epidermal growth factor in cultured bovine corneal endothelial and granulosa cells.
J. Biol. Chem
256
,
1149
1154
Shirota
M.
,
Banville
D.
,
Jolicoeur
C.
,
Boutin
J. M.
,
Edery
M.
,
Dijane
J.
,
Kelly
P. A.
(
1990
).
Expression of two forms of prolactin receptor in rat ovary and liver.
Mol. Endocrinol
4
,
1136
1144
Taniguchi
T.
(
1995
).
Cytokine signaling through nonreceptor protein tyrosine kinase.
Science
268
,
251
254
Wakao
H.
,
Gouilleux
F.
,
Groner
B.
(
1994
).
Mammary gland growth factor (MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response.
EMBO J
13
,
2182
2191
Walsh
R. J.
,
Posner
B. I.
,
Patel
B.
(
1984
).
Binding and uptake of 125I Iodoprolactin by epithelial cells of the rat choroid plexus: an in vivo autoradiographic analysis.
Endocrinology
114
,
1496
1505
Yeh
H. J.
,
Pierce
G. F.
,
Deuel
T. F.
(
1987
).
Ultrastructural localization of platelet-derived growth factor/v-sis-related protein(s) in cytoplasm and nucleus of simian sarcoma virus-transformed cells.
Proc. Nat. Acad. Sci. USA
84
,
2317
2321
This content is only available via PDF.