From the
The prolactin receptor (PRLR) belongs to the superfamily of
cytokine/growth hormone/prolactin receptors. Members of this family do
not contain a tyrosine kinase domain but are associated with
cytoplasmic kinases of the Jak family. Here, we examine different
mutants of the PRLR with respect to their ability to associate and
activate the kinase Jak2 and the transcription factor Stat1. Moreover,
using a biological assay system we are able to correlate these
activities with activation of prolactin-responsive gene transcription.
Our results indicate that interaction between Jak2 and PRLR requires a
proline-rich sequence in the membrane proximal region of the receptor,
which is conserved among the different members of the cytokine receptor
superfamily. We also show that association of Jak2 with the receptor is
sufficient for activation of the kinase as well as the transcription
factor Stat1. Moreover, our findings indicate that association of PRLR
with Jak2 is necessary but not sufficient for the transmission of a
lactogenic signal. We have identified two other cytoplasmic regions of
the PRLR that are required for activation of transcription. These two
regions are located between boxes 1 and 2 and are in the
carboxyl-terminal tail of the receptor. These sites probably involve
specific interactions with other effector molecules.
The prolactin receptor (PRLR)
Recently, efforts to identify signal
transducers activated by the cytokine/GH/PRL receptor family have
demonstrated that cytokine receptors associate with and activate
several cytoplasmic tyrosine kinases of the Janus tyrosine kinase
family
(3) . It has been shown for both the GHR and the PRLR
that this region including the first 85 amino acids (aa) and 94 aa of
the cytoplasmic domain, respectively, was also necessary for Jak2
association with these receptors
(4, 5) . In addition,
recent reports also showed for the EPOR and the
The cDNA encoding the PRLR has
been cloned from a number of different species
(8) and exists
in several natural forms. In the rat, two major forms have been
characterized: a short form of 291 aa with a cytoplasmic domain of 57
aa
(9) and a long form of 591 aa with a cytoplasmic region of
357 aa
(10) . These two forms share the same extracellular and
transmembrane domains and differ only in their carboxyl-terminal
cytoplasmic domain. In addition, an isoform of the rat PRLR has been
identified in the rat lymphoma cell line Nb2, representing a deletion
of 198 aa compared with the long form of the receptor
(11) .
Functional analysis of the three different natural forms of the PRLR
revealed that only the long and Nb2 forms are able to induce promotor
activity of the
Prolactin, similar to other cytokines, may
regulate gene transcription through activation of the newly identified
family of transcription factors designated as signal transducers and
activators of transcription (Stat)
(3) . Following their
phosphorylation, these homologous and heterologous multiprotein
complexes acquire the ability to translocate to the nucleus and induce
transcription of ligand-responsive genes. Recently, prolactin has been
shown to activate two members of this Stat family, Stat1, also known as
p91, and Stat5, also called mammary gland factor
(15, 16, 17) . The role of Stat5 in activation
of
In this report, we have studied the
molecular properties of the different forms of the PRLR as well as
several deletion and truncation mutants for their ability to associate
and activate the tyrosine kinase Jak2 and to undergo tyrosine
phosphorylation. The capacity of these receptor constructs to induce
prolactin-dependent transcriptional activation of the
In this paper, we have examined different natural and mutant
forms of the PRLR to determine regions of the receptor important for
association and activation of the kinase Jak2, receptor
phosphorylation, induction of the
Our studies reveal that the
natural forms of the PRLR, including the short form, are able not only
to associate with the kinase but also to activate its phosphorylation,
suggesting that for this receptor, box 1 is sufficient for Jak2
association. To confirm this finding and extend it to the long and Nb2
forms of the PRLR, we prepared several deletion mutants of the
cytoplasmic domain of the PRLR in the juxtamembrane domain and tested
their ability to associate and activate the tyrosine kinase Jak2. Only
the deletion of box 1 prevents Jak2 association with the PRLR. In
contrast, a deletion between boxes 1 and 2 has no effect on the ability
of the receptor to interact with the kinase, a result which differs to
that obtained for EPOR and GM-CSFR
(6, 7) . It is
possible that association of Jak2 with some receptors involves
different cytoplasmic regions, in addition to the conserved box 1
motif. Interestingly, box 1 resembles an SH3 binding site
(2) ,
yet Jak2 does not contain an SH3 domain. This raises the possibility of
an adapter molecule being required to interact with box 1 through a
potential SH3 domain and bind Jak2 through a phosphotyrosine-SH2 domain
interaction. However, this hypothesis remains to be verified and does
not exclude other potential sites of interaction.
We observed PRLR
tyrosine phosphorylation in Nb2 cells following prolactin stimulation
(19) . Here, we demonstrate that activation of this process
requires the membrane proximal region, box 1, and the membrane distal
region common to the PRLR long and Nb2 forms. These results suggest
that Jak2 activation is not sufficient to induce receptor
phosphorylation. The carboxyl-terminal region of the receptor may
possess sites capable of interacting with another protein tyrosine
kinase. Alternatively, this carboxyl-terminal domain may harbor
tyrosine residues that undergo phosphorylation in response to prolactin
activation.
To correlate Jak2 association and activation and
receptor phosphorylation with the biological activity of the PRLR, we
developed a functional assay by coexpressing cDNAs encoding different
forms of the PRLR with an expression vector containing the
Interestingly, the
generation of mitogenic signals by other members of the superfamily of
cytokine receptors seems to not require carboxyl-terminal domain
sequences, as shown for the signal transducer gp 130
(22, 23) , granulocyte-colony stimulating factor
receptor
(24, 25) , IL-2R
Transcriptional activation of
cytokine and growth factor-responsive genes are mediated in part
through tyrosine phosphorylation of Stat proteins. Stat1 is tyrosine
phosphorylated in response to several cytokines including prolactin
(3, 15) . Cotransfection of Stat1 cDNA with cDNAs
encoding different forms of PRLR reveal that phosphorylation of Stat1
requires Jak2 association with the PRLR and does not appear to involve
other cytoplasmic domains of the receptor. When increasing amounts of
cDNAs encoding Jak2 and for Stat1 were transfected, a significant
increase in
Together, these
results demonstrate that the tyrosine kinase Jak2 associates with the
PRLR, a member of the cytokine/GH/PRL receptor family, through a
proline-rich motif in the membrane proximal region, and that the
interaction of the kinase with the receptor is necessary for activation
of phosphorylation of the transcription factor Stat1 but not sufficient
for signal transduction. Generation of a lactogenic signal further
requires the association of the receptor with one or more signaling
molecules within the cytoplasmic domain of the PRLR, which are likely
to be necessary for coupling to specific signal-transducing proteins.
We are grateful to Dr. J. Ihle for kindly providing
the cDNA encoding for the tyrosine kinase Jak2, to Dr. R. Lammers for
the antibody anti-Stat1 and for cloning the Stat1 cDNA, to Dr. J. Rosen
for providing the rat
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
belongs to
the recently described superfamily of cytokine/GH/PRL receptors
(1) , which share several structural features, including a
unique transmembrane domain, an extracellular region containing four
conserved cysteine residues, and a WS XWS motif. The
cytoplasmic domains of these receptors, which contain no intrinsic
kinase function, show very low overall sequence identity. The membrane
proximal region (100 amino acids) of the receptors from this
superfamily includes two conserved motifs: box 1, which contains a
proline-rich domain, known to be involved in protein-protein
interactions
(2) , and box 2, which does not contain any known
consensus sequence. It has been shown for many members of the family
that mutations in this membrane proximal region result in a loss of
functional activity, which suggest that it plays a crucial role in
signal transduction.
-chain of the
GM-CSFR that the region between the two boxes was necessary for the
interaction between the kinase and the receptor
(6, 7) .
These data indicate that the site of interaction between the tyrosine
kinases from the Jak family and the cytokine receptors is located in
this extended juxtamembrane region.
-casein promotor
(12, 13) as well
as the prolactin-inducible T cell activation gene, interferon
regulatory factor-1
(14) . These results suggest that the
cytoplasmic regions common to the long and Nb2 forms are sufficient for
signal transduction.
-casein gene transcription has recently been examined
(17) . However, the role of Stat1 activation in this signaling
pathway remains unknown.
-casein gene
promotor and to activate a transcription factor Stat1, known to be
involved in PRLR signaling, was also examined. Our results indicate
that the PRLR-associated kinase Jak2 binds to the proline-rich region
of the receptor and that this interaction is absolutely necessary for
tyrosine phosphorylation of the kinase, the receptor, and the
transcription factor Stat1. However, our results also demonstrate that
these activities are not sufficient for prolactin-mediated
transcriptional activation of the
-casein gene promotor. Finally,
we have characterized two other regions of the PRLR cytoplasmic domain
including the region between box 1 and box 2 and the carboxyl-terminal
tail of the receptor to be necessary for activation of milk protein
gene transcription.
Materials
Ovine prolactin (oPRL)
(NIDDK) was obtained from the National Hormone and Pituitary
program/NIDDK (Baltimore, MD). Recombinant bovine GH was kindly
provided by Dr. W. Brumbach (American Cyanamid Co., Princeton, NJ) (ref
9450-1-7).
Cell Culture and Transfection
The 293
fibroblast cells and the stable clone expressing the tyrosine kinase
Jak2 (clone LA) were grown in DMEM nut F12 medium containing 10% fetal
calf serum. Several hours before transfection, cells were plated in a
rich medium ( DMEM nut F12, DMEM 4.5 g/liter glucose) containing 10%
fetal calf serum. Then, the cells (5 10
) were
transfected with the indicated amounts of cDNAs encoding Jak2 (kindly
provided by Dr. J. Ihle), Stat1 (cloned from a human placenta library),
and the different forms of PRLR. After 24 h of expression, the cells
were deprived of serum for an overnight period.
Establishment of a 293 Cell Line Stably Expressing
the Tyrosine Kinase Jak2 (Clone LA)
293 cells at 50%
confluency were cotransfected with an expression vector encoding the
tyrosine kinase Jak2 and an expression vector (pSVneo) encoding the
resistance gene neomycine (Pharmacia Biotech Inc.) at a 10/1 ratio
using the calcium phosphate technique. 2 days following transfection,
medium was changed, and fresh medium containing G418 at 500 µg/ml
was added; after three weeks, 70 resistant colonies were selected, and
Jak2 expression in these cells was examined by Western blotting with a
polyclonal anti-Jak2 antibody (UBI). We selected several clones
expressing different levels of the kinase Jak2 and used the LA clone in
the present study, which expresses a high level of Jak2.
Cell Surface Labeling of the PRLR
The
PRLRs expressed at the cell surface were labeled by incubation with a
monovalent Fab fragment generated from a monoclonal antibody (U5)
directed against the extracellular domain of the PRLR at 0.5 µg/ml
for 20 min at 37 °C before stimulation.
Purification of the PRLR Complexes
Cells
were stimulated with either oPRL or biotinylated oPRL and recombinant
bovine GH for 10 min at 5 10
M.
Then, the cells were lysed in 1 ml of lysis buffer (10 mM
Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 30
mM sodium pyrophosphate, 50 mM sodium fluoride, 1
mM sodium orthovanadate, 10% glycerol, 0.5% Triton X-100)
containing protease inhibitors (1 mM phenylmethylsulfonyl
fluoride, 1 µg/ml pepstatin A, 2 µg/ml leupeptin, 5 µg/ml
aprotinin) for 10 min at 4 °C. Then, the insoluble material was
discarded by centrifugation at 12,000
g for 5 min, and
the amount of protein was equalized in all the samples by a protein
concentration measurement using the Bradford technique. PRLR complexes
were then incubated with 10 µl of avidin-agarose beads (10% w/v in
lysis buffer) for 2 h at room temperature for the samples stimulated by
biotinylated hormones or with 10 µl of anti-IgG-agarose beads (10%
w/v in lysis buffer) at 4 °C for the samples labeled with the Fab
monovalent fragment. Samples were washed three times in 1 ml of lysis
buffer and eluted in 20 µl of SDS loading buffer (20% glycerol, 10%
-mercaptoethanol, 4.6% SDS, 0.125 M Tris-HCl, pH 6.8).
Immunoprecipitation with an Anti-Stat1
Antibody
Cell lysates were immunoprecipitated for 3 h at 4
°C with 2 µl of Stat1 polyclonal antibody raised against the
peptide CPEEFDEVSRIVGSVEFD and 10 µl of protein A-Sepharose beads
before being washed and eluted as described above.
Western Blot Analysis
Proteins were
separated on a 7.5% acrylamide gel, transferred onto nitrocellulose,
and immunodetected with either an anti-phosphotyrosine antibody (4G10;
Upstate Biotechnology, 0.1 µg/ml) or an anti-PRLR monoclonal
antibody (U5 at 0.5 µg/ml) for an overnight period at 4 °C.
Then, the membranes were incubated with a anti-mouse antibody coupled
to peroxidase for 1 h at room temperature before being washed four
times for 30 min in the washing buffer (50 mM Tris-HCl, pH
7.6, 200 mM NaCl, 0.05% Tween 20) and revealed by
chemioluminescence (ECL kit from Amersham Corp.) according to the
manufacturer's instructions. Membranes were stripped overnight at
4 °C in an acid solution (0.1 M glycine, pH 3, 0.1
M NaCl) and reprobed with an anti-Jak2 antibody (UBI, dilution
1/4000) and then with an anti-rabbit antibody coupled to peroxidase
before being processed as described above.
Mutagenesis Experiments
The construction
of the deletion mutants ( 243-267) and (
296-322), as well as the truncation mutant (T322) were carried
out using the Nb2 PRLR cDNA subcloned in M13 mp18 bacteriophage as a
template. Single-stranded DNA was generated for the
oligonucleotide-directed mutagenesis in RZ 1032 cells. After being
checked by sequencing, the modified DNA were subcloned into the
eukaryotic expression vector pR/CMV. The construction of the (
268-287) mutant was carried out directly into the expression
vector pR/CMV, using its M13 origin of replication and the MKO7 M13
helper phage. Single-stranded DNA was generated for mutagenesis
reactions in CJ236 cells. Mutants were checked by DNA sequencing.
Prolactin-dependent Induction of
Cells were
plated in 6-well plates (0.5 -Casein
Promotor/Luciferase Construct in 293 Cells
10
cells/well) before
being transiently cotransfected as described before with 0.5 µg of
pCH110 (
-galactosidase expression vector, Pharmacia), 0.2 µg
of the fusion gene carrying the promotor region of the rat
-casein
gene linked to the coding region of the luciferase gene and 0.1 µg
of plasmid pR/CMV/long, pR/CMV/short, pR/CMV/Nb2, pR/CMV/
243-267, pR/CMV/
268-287, pR/CMV/
296-322,
and pR/CMV/T322. 1 day after transfection, cells were incubated in the
presence or the absence of human PRL (5
10
M) and dexamethasone (250 nM) for 24 h before
being lysed. Each value represents the stimulated luciferase activity
measured in relative light units and normalized for
-galactosidase
activity. Results are the means ± S.E. of four different
experiments.
Cotransfection of the cDNAs Encoding the Tyrosine
Kinase Jak2 and the PRLR
To evaluate the association
between the PRLR and Jak2, we have used transient expression in human
293 fibroblasts. Fig. 1shows the optimal conditions required for
a clear induction of Jak2, and PRLR phosphorylation occurs with low but
equal concentrations of each cDNA. Increasing the concentrations of
cDNA for Jak2 results in its overexpression and nonspecific
phosphorylation in the absence of ligand. Nevertheless, ligand-induced
phosphorylation can still be observed, but the degree of induction is
reduced. Increasing the amount of PRLR cDNA transfected also led to a
high nonspecific signal in absence of prolactin. These results suggest
that in 293 cells, it is extremely important to control the level of
expression of both receptor and kinase to see clearly ligand-stimulated
phosphorylation. If either receptor or kinase is overexpressed,
autophosphorylation of both occurs in the absence of prolactin, perhaps
due to an excess of kinase or to non-ligand-induced dimerization.
Figure 1:
Specificity of ligand-induced
phosphorylation of Jak2 and the PRLR. 293 fibroblasts (2.5
10
/lane) were transfected with the indicated amounts of
cDNAs encoding Jak2 and long form of PRLR. Prolactin receptors
expressed at the cell surface were labeled with a monovalent Fab
fragment generated from a monoclonal antibody (U5) directed against the
extracellular domain of the PRLR, and the PRLR complexes were purified
by incubation with anti-IgG-agarose beads. Proteins were separated on a
7.5% acrylamide gel, transferred onto nitrocellulose, and hybridized
with an anti-phosphotyrosine antibody (4G10; Upstate Biotechnology) and
revealed by ECL (Amersham), according to the manufacturer's
instructions. The size of the two phosphorylated proteins,
corresponding to the PRLR and the Jak2 kinase, are 95 and 130 kDa,
respectively, and are indicated to the
right.
Association and Activation of the Tyrosine Kinase
Jak2 with the Different Natural Forms of the PRLR
The Nb2
form of the PRLR is a natural mutant of the long form of the receptor
(Fig. 2) and has been shown, as well as the long form, to be
constitutively associated with the tyrosine kinase Jak2
(18, 19, 20, 21) . We were interested in
determining whether the short form was also able to bind and activate
Jak2 as well as to undergo tyrosine phosphorylation itself.
Fig. 3
, A and B, show that coexpression of the
long or Nb2 forms of PRLR with Jak2 in human 293 fibroblasts results in
association of the kinase with each form of PRLR and in tyrosine
phosphorylation of both receptor and kinase. This indicates that PRLR
binding and activation of the Jak2 kinase are key events in the
transduction of the prolactin signal. Moreover, the deleted sequences
within the cytoplasmic domain of the Nb2 form (Fig. 2) appear not
to be necessary for Jak2 association. Interestingly, the short form was
also able to bind and activate Jak2 but not undergo tyrosine
phosphorylation, despite a high level of receptor expression
(Fig. 3 C). This indicates that only the residues common
to all three PRLR isoforms are required for Jak2 interaction. Moreover,
our results also indicate that receptor phosphorylation occurs only in
cytoplasmic regions present on PRLR long and Nb2 forms. This is
especially important, since the short form of the PRLR is unable to
generate a lactogenic signal
(12, 13) nor to induce
interferon regulatory factor-1 transcription
(14) . This leads
to the speculation that in addition to Jak2 phosphorylation, PRLR
tyrosine phosphorylation is also required for prolactin signal
transduction. An anti-Jak2 immunoblot was performed after cell surface
labeling of the PRLR with a monovalent Fab fragment generated from an
anti-PRLR monoclonal antibody (U5), as described in Fig. 1, and
revealed no change in the amount of Jak2 bound to the short form of the
PRLR in the absence or presence of prolactin (data not shown). This
indicates that the short form of the PRLR is constitutively bound to
the kinase Jak2, as is true for the long and Nb2 forms.
Figure 2:
Schematic representation of wild type and
mutant forms of PRLR. Illustrated are wild type forms of the rat long,
Nb2, and short PRLR, as well as the mutant forms established from the
Nb2 PRLR 243-267,
268-287,
296-267,
and T322. Transmembrane domains are represented by black
boxes. The juxtamembrane region conserved between GH and PRL
receptors, containing the box 1 proline-rich motif, is
stippled. Numbers to the right indicate the
first amino acid, and the last amino acid of the mature protein. For
the deletion mutants, the actual number is 393 minus the
number of residues removed.
Figure 3:
Association and activation of the tyrosine
kinase Jak2 with the different forms of PRLR in response to prolactin.
A, tyrosine phosphorylation of the kinase and the receptor.
Human 293 fibroblasts were cotransfected with the calcium phosphate
technique, with 2.5 µg of cDNA encoding the different natural forms
of PRLR (long, short, or Nb2) and 2.5 µg of cDNA encoding the human
tyrosine kinase Jak2. After stimulation by either the non-lactogenic
biotinylated recombinant bovine GH or the lactogenic biotinylated oPRL,
cell lysates were processed as described under ``Experimental
Procedures'' and immunoblotted with a monoclonal
anti-phosphotyrosine antibody. The position and the size of Jak2 (130
kDa) is indicated to the right as well as the size of the two
forms of PRLR, which are tyrosine phosphorylated in response to
prolactin, the long form (95 kDa), and the Nb2 form (62 kDa).
B, Jak2 association with the different natural forms of the
PRLR. The membrane described in A was stripped and reprobed
with an anti-Jak2 antibody. The size of Jak2 is indicated on the
right (130 kDa). C, to verify the correct expression
of all the forms of PRLR, the membrane used in A was stripped
and reprobed with a monoclonal anti-PRLR antibody. The size of the
natural forms of the PRLR are 95, 62, and 42 kDa for the long, Nb2, and
short forms, respectively.
Box 1 Is Sufficient for Jak2 Association and
Activation
Since only box 1 is conserved in all three
natural forms of the PRLR (Fig. 2), this led us to evaluate the
importance of this region, as well as other regions of the cytoplasmic
domain of the PRLR, for Jak2 association. We constructed PRLR mutants
containing a deletion of 20 aa comprising the proline-rich domain
( 243-267), a deletion of 19 aa (
268-287)
corresponding to the region between the two boxes, or a control
deletion of 27 aa, including part of the box 2 domain (
296-322). Finally, we constructed a truncated mutant of the PRLR
(T322), in which the carboxyl-terminal 70 aa, common to the long and
Nb2 forms, were deleted (Fig. 2). Specific binding of
I oPRL to microsomes prepared from transfected 293 cells,
as well as whole cell binding, revealed that all wild and mutant forms
of the receptor were expressed at approximately equal levels (data not
shown). Immunoblot analysis of extracts from cells coexpressing the
different mutants and Jak2 demonstrated that
268-287,
296-322, and T322 but not
243-267 were able to bind
(Fig. 4, C and D) and induce tyrosine
phosphorylation of Jak2 (Fig. 4, A and B). The
cell surface labeling of these mutant forms expressed in 293 cells with
the Fab fragment and the immunoblot analysis with an anti-Jak2
antibody, as described in the Fig. 1legend, also revealed that
all the mutant forms of the PRLR, except
243-267, were
constitutively bound to the kinase Jak2 (data not shown). These data,
in addition to the previous results obtained with the short form of the
PRLR, strongly suggest that the proline-rich region is the site of
interaction with Jak2, implicating this sequence as being essential for
signal transduction. Other parts of the juxtamembrane domain, as well
as the carboxyl-terminal tail of the receptor, affect neither
association of the receptor with Jak2 nor tyrosine phosphorylation of
the kinase.
Figure 4:
Association and activation of the tyrosine
kinase Jak2 with different mutant forms of PRLR in response to
prolactin. A and B, in this experiment, cells were
cotransfected with the cDNA encoding Jak2 and one of the four mutant
forms of the Nb2 PRLR (T322, 243-267,
296-322,
and
268-287 compared with the wild type Nb2 PRLR). The
immunoblot analysis was performed with an anti-phosphotyrosine
antibody. The size of Jak2 (130 kDa) and the mutants
243-267 (59 kDa),
296-322 (58 kDa),
268-287 (59 kDa), and the wild type Nb2 PRLR are indicated to the
right. C and D, Jak2 association with the
mutant forms of the PRLR. The previous blots ( A and
B) were stripped and reprobed with an anti-Jak2 antibody
(UBI). E and F, the correct expression of the mutant
forms of the PRLR was verified by reprobing of the membrane with a
monoclonal antibody against PRLR. The sizes are 50, 58, 58, 59, and 62
kDa for T322,
243-267,
268-287,
296-322, and the wild type Nb2 PRLR,
respectively.
We have previously shown in Nb2 cells that upon
prolactin stimulation, the PRLR and Jak2 undergo tyrosine
phosphorylation
(19) . Examining receptor phosphorylation, shown
in Fig. 4, A and B, indicates that the deletion
mutant ( 243-267) lacking box 1 domain and the truncation
mutant (T322) failed to induce receptor phosphorylation, while the
deletion mutants
268-287 and
296-322 were able
to undergo tyrosine phosphorylation themselves. These results further
demonstrate that the site of interaction of Jak2 with the PRLR is
mediated by box 1 only and that receptor phosphorylation requires the
proper receptor-kinase association. The proper expression of these
mutants has been verified by Western blot, as shown in Fig. 4,
E and F. Since this proline-rich motif is found in
many members of the cytokine receptor superfamily, it is likely that
other members of the cytokine/GH/PRL family also interact with Jak2
through the same conserved domain (Fig. 5).
Figure 5:
Amino acid sequences conserved in the
cytoplasmic domains of members of the cytokine/GH/PRL receptor
superfamily. The proline-rich region of the PRLR, GHR, IL-6R, gp 130,
EPOR, granulocyte-colony stimulating factor receptor, and GM-CSFR
subunits common to IL-3R, IL-5R, IL-2R
, IL-3R, IL-4R, IL-7R,
IL-5R, GM-CSFR, leukemia inhibitory factor receptor, and
myeloproliferative leukemia virus receptor are shown. Amino acids
highlighted in bold represent those identical to the PRLR.
Numbers in parentheses indicate the beginning and end
of the proline-rich region; the first amino acid (1) corresponds to the
initiator methionine.
Regions of the Cytoplasmic Domain Required for
Activation of
To correlate these
findings with the ability of the receptor to transmit a lactogenic
signal, we cotransfected the different natural and mutant forms of the
PRLR, described above, with the -Casein Gene Promotor
-casein promotor coupled to the
luciferase gene in 293 cells, as described under ``Experimental
Procedures.'' The results indicate that similar to the short form,
the deletion mutants
243-267 and
268-287 as
well as the truncation mutant T322 failed to activate the
-casein
promotor (Fig. 6). However, the deletion mutant
296-322 was fully capable of inducing
-casein promotor to a
similar level to that seen with PRLR long and Nb2 forms (Fig. 6).
These observations indicate that three complementary mechanisms are
needed for achieving full biological signaling of the PRLR. These
include association of the kinase Jak2 with the PRLR mediated through
box 1, the interbox region, as well as the carboxyl-terminal part of
the receptor. The last two regions possibly present sites of
interaction with other transducer molecules involved in prolactin
signaling pathway.
Figure 6:
Prolactin-dependent induction of
-casein promotor/luciferase construct in 293 cells. Cells were
transfected as described in under ``Experimental Procedures''
with the expression vectors containing the cDNAs encoding the different
forms of PRLR. Results are expressed as the percentage of maximal
activity (-fold induction). Results represent means ± S.E. of
four independent experiments. The leftpanel represents the natural and mutant forms of the PRLR; the
numbers to the right indicate the last amino acid of the
mature protein. For the deletion mutants, the actual number is 393
minus the number of residues removed.
Activation of the Transcription Factor
Stat1
Prolactin has been shown to activate two
transcription factors belonging to the Stat family, Stat1 and Stat5.
Stat1 has also been shown to be activated in response to different
cytokines and to bind responsive elements related to the palindromic
sequence recognized by Stat5. Stat5 activation has been shown to be
indispensable for the hormonal induction of the -casein gene
transcription. Therefore, we were interested in correlating the results
of transcriptional activation described above with the ability of the
different forms of PRLR to activate Stat1. For that purpose, we
cotransfected a 293-cell line stably expressing the tyrosine kinase
Jak2 with the cDNAs encoding Stat1 and a cDNA specific to the different
forms of PRLR. As shown in Fig. 7 A, tyrosine
phosphorylation of Stat1 is seen with all natural and mutant forms of
PRLR, except
243-267, in which box 1 has been deleted. This
suggests that activation of Stat1 depends on Jak2 and requires a
functional kinase molecule bound to the receptor. In addition,
activation of Stat1 seems to not require other regions of the
cytoplasmic domain of the PRLR, since all other forms of the PRLR are
able to induce Stat1 phosphorylation. When the membrane was stripped
and reprobed with an antibody directed against Stat1, it is clear that
the Stat protein is expressed equally in all samples
(Fig. 7 B). The absence of clear induction in the
phosphorylation of Stat1 upon prolactin stimulation is probably due to
the high level of expression of the receptor, resulting in
ligand-independent receptor dimerization and constitutive activation of
the kinase Jak2. We confirmed this by cotransfecting the cDNAs encoding
the long and short forms of the PRL receptor at different
concentrations, with the cDNA encoding Stat1, in this cell line and
obtained a clear prolactin induction of Stat1 phosphorylation by
decreasing the amount of PRLR cDNA (data not shown). For Stat1
activation, Jak2 must be associated with the PRLR, as shown by the
absence of signal when Stat1 is transfected alone in the stable cell
line expressing Jak2. Since Stat1 activation is also mediated through
cytokine receptors that are able to activate the tyrosine kinase Jak1,
we examined the ability of the long form of the PRLR to induce tyrosine
phosphorylation of Jak1 in our system. No Jak1 phosphorylation was
observed following expression of PRLR in 293-LA cells (data not shown).
This is in agreement with what we observed previously in Nb2 cells
(19) .
Figure 7:
A, activation of tyrosine phosphorylation
of the transcription factor Stat1 with the different forms of PRLR in
response to prolactin. The 293 stable cell line expressing the tyrosine
kinase Jak2 was cotransfected with the cDNA encoding the different
natural and mutant forms of PRLR and the cDNA encoding the Stat1
molecule. After stimulation by oPRL, cell lysates were
immunoprecipitated with an anti-Stat1 antibody and immunoblotted with a
monoclonal anti-phosphotyrosine antibody. The position and the size of
Stat1 (91 kDa) and of the heavy chains of IgG (55 kDa) are indicated to
the right. B, to verify the correct expression of
Stat1 in all the assays, the membrane was stripped and immunodetected
with an anti-Stat1 monoclonal antibody (Transduction Laboratories). The
size of Stat1 (91 kDa) and of the heavy chains (55 kDa) are indicated
to the right.
-casein gene promotor, and
activation of the transcription factor Stat1. For this purpose, we
developed a transient cotransfection system in which both the kinase
Jak2 and the different forms of the PRLR were expressed in human kidney
fibroblast 293 cell line. To see a clear result, it is important to
determine the amount and ratio of each cDNA transfected. The fact that
Jak2 is phosphorylated even in absence of prolactin stimulation could
be due to an autoactivation of the kinase due of overexpression, but it
also could reveal a normal state of phosphorylation, amplified in our
system by the high number of copies of the kinase. In fact, it is
clearly possible that some tyrosine residues of the kinase are
phosphorylated, even in absence of stimulation, especially as the
association between the kinase Jak2 and the PRLR has been shown to be
constitutive
(18, 19, 21) . Such
phosphotyrosines, present in the kinase, could interact with the SH2
domain of an adaptor molecule, which in turn could interact with the
receptor through another SH2 or an SH3 domain. Such a molecule, if it
exists, remains to be identified.
-casein
promotor coupled to the luciferase gene. Utilizing this system, our
results demonstrate that Jak2 association with the PRLR is necessary
but not sufficient for activation of transcription. Thus, in addition
to Jak2 association involving box 1, activation of milk protein gene
transcription requires the presence of other residues in the membrane
proximal region between boxes 1 and 2, as well as in the
carboxyl-terminal part of the cytoplasmic domain. These results are in
agreement with other observations, indicating that several regions,
including box 1 and box 2, were required for the transmission of the
growth signal by prolactin
(5) .
(26) , EPOR
(27, 28, 29) , and the GHR
(30) . However
for the GHR, a recent report indicated that the carboxyl-terminal
portion of the receptor is required for activation of transcription
(31) . In addition, it has been shown that the carboxyl-terminal
region of the cytoplasmic domain of the IFN
was also necessary for
activation of the IFN
regulatory factor 1 gene
(32) . For
the long form of the PRLR from rabbit mammary gland, deletion of the
carboxyl-terminal 70 aa has no effect on lactogenic signal
transduction, but further deletion by another 70 aa into the region
present in the Nb2 form results in loss of transcriptional signaling
activity
(33) . This suggests that the long form of the PRLR
contains another site of interaction with the same or another
transducer molecule of the prolactin signaling pathway and that the
deletion of the carboxyl-terminal domain alone does not necessarily
result in the loss the activity.
-casein-luciferase activity was observed in the
absence or presence of ligand but without changing the overall fold
induction (data not shown). This indicates that the Jak2/Stat1 pathway
may be involved in the
-casein promotor regulation but is not
sufficient for its activation. Since another member of the Stat family,
Stat5, is also involved in the regulation of this promotor, it is
possible that the complete and active transcription factor, which binds
to the
-casein promotor, could in fact be a homo- or heterodimer
of Stat molecules, as it has been demonstrated for other receptors from
the cytokine family. Tyrosine phosphorylation of Stat5 in response to
prolactin has been shown to be mediated by the long form of the PRLR
while the short form failed to transmit this effect
(17) . This
indicates that the mechanisms involved in activation of these two Stat
proteins are different. It is possible that Stat1 activation is
mediated through Jak2 activity, while Stat5 activation requires either
direct interaction of the molecule with sequences present in the
cytoplasmic domain of the long form of PRLR or indirect interaction,
requiring the presence of other signaling molecules. Mutational
analysis of the tyrosine residues present in Jak2 and PRLR are being
carried out to investigate these possibilities.
,
subunit of the interleukin-2 receptor;
GHR, growth hormone receptor; GM-CSFR, granulocyte
macrophage-colony-stimulating factor receptor; gp 130,
-subunit of
the IL-6R, leukemia inhibitory factor receptor; OSMR, oncostatin M
receptor; CNTFR, ciliary neurotrophic factor receptor; EPOR,
erythropoietin receptor; GM-CSFR
, common
-subunit of the
GM-CSFR, IL-3R, and IL-5R; aa, amino acids; DMEM, Dulbecco's
modified Eagle's medium.
-casein promoter, and to H. Buteau for
preparing the
-casein/luciferase construct, to Dr. J. Finidori for
subcloning the Jak2 cDNA into the expression vector pCB6, to the
National Hormone and Pituitary program for providing ovine prolactin,
and to Dr. W. Brumbach for the recombinant bovine GH.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.