The Short Form of The Prolactin (PRL) Receptor Silences PRL Induction of the ß-Casein Gene Promoter
Juan José Berlanga1,
Josefa P. Garcia-Ruiz,
Martine Perrot-Applanat,
Paul A. Kelly and
Marc Edery
Institut National de la Santé et de la Recherche
Médicale, Unité 344 Endocrinologie Moléculaire
(J.J.B; M.P-A; P.A.K; M.E.), Faculté de Médecine
Necker, 75730 Paris Cedex 15, France,
Departamento de
Biologia Molecular-Centro de Biologia Molecular Severo Ochoa,
Universidad Autonoma de Madrid, 28049 Madrid, Spain
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ABSTRACT
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The PRL receptor (PRLR) is a member of the
cytokine receptor superfamily. Rats and mice express two forms of PRLR,
short (SPRLR) and long (LPRLR), which differ in the length and sequence
of their cytoplasmic domains. We have analyzed the ability of each form
of rat PRLR to transduce lactogenic signals in a bovine mammary gland
epithelial cell line. The rat PRLR forms were expressed and detected by
RT-PCR, indirect immunofluorescence, and cell surface ligand binding.
When the biological activity of each form of PRLR was assessed by
transient transfection, we found that the long form was able to
activate the ß-casein gene promoter and that the short form was
inactive. Interestingly, the coexpression of both forms of PRLR
resulted in a block of PRL signal to the milk protein gene promoter as
a function of the concentration of the SPRLR. Similar results were
obtained when LPRLR was coexpressed with totally or partially inactive
tyrosine mutants of either the Nb2 form or the LPRLR form. Thus, these
results suggest that the SPRLR form has at least one clear biological
function, i.e. to silence lactogenic signals and to
contribute to a differential and acute PRL effect in rat tissues.
Furthermore, the data derived from coexpression of LPRLR and PRLR
mutants confirm a crucial role of the C-terminal tyrosine residue in
lactogenic signaling and the dimerization of PRLRs.
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INTRODUCTION
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The pituitary hormone PRL has been involved in a variety of
biological functions, including the synthesis of milk proteins, immune
responses, development of reproductive organs, osmotic balance, and
behavior. The PRL receptor (PRLR) has been characterized from different
species, and it is well documented that, at least in rats and mice, it
is expressed as short (SPRLR) and long (LPRLR) receptor isoforms (1).
These PRLR isoforms are proteins coded by a single gene and generated
by alternative splicing (2). As a result, the two forms of PRLR only
differ in the amino acid composition and size of the polypeptide chain
beyond the first 27 amino acids of their respective intracellular
domains. The transcripts of both forms are variably expressed in a
tissue-specific manner, depending on the stage of the estrous cycle,
pregnancy, and lactation (3, 4).
PRLRs are members of the cytokine family of receptors (1). Ligand
activation of this type of receptor triggers the activation of
cytoplasmic protein tyrosine kinases (5). In the case of rat PRLRs, it
has been demonstrated that protein tyrosine kinases of the Jak and Src
families are associated with PRLRs and are involved in the
intracellular signaling of PRL (6, 7, 8, 9, 10). Functional analysis of these
receptors has shown that LPRLR, as well as a third form, the Nb2PRLR,
characterized in the rat lymphoma cell line Nb2 (11), are able to
transduce PRL signals to the milk protein and interferon regulatory
factor-1 promoters (12, 13). However, because SPRLR was inactive in the
functional studies mentioned above, its function remains unclear. A
recent report showed that the SPRLR is functional, being able to
stimulate NIH 3T3 cell proliferation, an action that is preceded by
transient activation of mitogen-activating protein kinase, while it is
unable to activate the ß-casein gene promoter (14). The SPRLR shares
with the LPRLR the first 27 intracellular amino acids that contain a
proline-rich region (Box 1) involved in the association with the
tyrosine kinase Jak2 (15). This tyrosine kinase has been shown to
associate with both forms of PRLR and to play an important role in the
intracellular signaling of PRL (6, 7, 8). In addition, both PRLR isoforms
associate with the tyrosine kinase p59fyn (9) or the
cytosolic protein tyrosine phosphatase PTP1D in Nb2 cells (16).
In this study, we assessed the biological function of the two rat PRLR
isoforms by expressing them in a bovine mammary gland epithelial cell
line (BMGE). When rat PRLRs were expressed using the recombinant
vaccinia-T7 virus expression system, both forms were shown to be
present as clusters in mammary epithelial cells. PRL signaling through
each PRLR isoform was assayed by transient cotransfection with a vector
containing ß-casein gene promoter. Our results show that the long
form mediates PRL activation of a milk protein gene promoter while the
short form is inactive, in agreement with earlier data (12). However,
we found that cotransfection of cDNA from both PRLR isoforms blocked
the signaling of LPRLR to milk protein gene promoter. Thus, our data
strongly suggest that the short form of PRLR has a silencing role in
PRL signaling to milk protein production. In addition, signaling of the
LPRLR can be blocked when it is cotransfected with inactive PRLR
tyrosine mutants. The silencing role of the SPRLR may have
physiological significance because the expression of the two forms of
PRLR is regulated in a tissue-specific manner.
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RESULTS
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PRLR Expression in Transfected BMGE Cells
To determine whether BMGE cells could be used to study the
functional activity of rat PRLR isoforms, we first tested the
expression of these proteins in BMGE cells by using a recombinant
vaccinia-T7 virus expression system that allows a high level of
expression. After infection with vaccinia-T7, cells were transfected
with either pTM1-LPRLR or pTM1-SPRLR constructs. The receptor expressed
was detected by indirect immunofluorescence using the anti-PRLR T1
antibody (17). As can be observed in Fig. 1
, both PRLR forms were expressed in BMGE
cells. The presence of immunofluorescent clusters can be attributed to
the accumulation of overexpressed PRLR using the vaccinia-T7 virus
system or to fixation conditions.

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Figure 1. Expression and Cellular Localization of Short and
Long PRLR Forms in BMGE Transfected Cells
Cells were infected with vaccinia-T7 virus and then transfected with
pTM1-LPRLR or pTM1-SPRLR constructs. At 14 h posttransfection, the
cells were fixed and PRLR isoforms were detected by indirect
immunofluorescence using T1 monoclonal antibody. A, Vaccinia-T7 virus
infected, but untransfected, cells. B, Cells transfected with
pTM1-LPRLR. C, Cells transfected with pTM1-SPRLR. D, Cells transfected
with pTM1 LPRLR and pTM1 SPRLR.
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To further confirm the presence of PRLR, Flag-tagged PRLR cDNA (18) was
transfected in BMGE cells using the calcium phosphate method; cells
were subsequently incubated in the presence (or not) of PRL and treated
with anti-Flag antibodies, as previously described (18). In the absence
of hormone, immunofluorescence for Flag-PRLR was observed on the
cell surface in nonpermeabilizing conditions (Fig. 2a
) and intracellularly (not shown);
stimulation with ovine (o)PRL led to the disappearance of
immunofluorescence from the plasma membrane as visualized using
nonpermeabilizing conditions (not shown) and presence within the
cytoplasm (bright dots), clearly observed using permeabilizing
conditions (Fig. 2b
); such localization probably corresponds to
vesicles of the endocytic pathway, as previously described in detail in
several other cell types (18). Control experiments in which cells were
untransfected showed no background staining (Fig. 1A
and Fig. 2c
).

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Figure 2. Indirect Immunofluorescence Localization of
Flag-tagged PRLR Expressed in BMGE Cells
BMGE cells transfected with cDNAs (5 µg) encoding a flag-tagged
PRLR were deprived from serum overnight and fixed at 10 min8 h after
oPRL (400 ng/ml) addition (b, 20-min incubation) or without hormone (a,
control), as described in Materials and Methods; control
untransfected cells were also used (c). Fixation with 4%
paraformaldehyde and permeabilization with methanol (-20 C) were used
in panels b and c, while fixation without or with permeabilization was
used to localize PRLR at the cell surface membrane or intracellularly,
respectively, for both hormonal conditions. After fixation, cells were
immunostained using a mouse monoclonal anti-flag antibody (10 µg/ml)
as primary antibody and fluorescein-conjugated anti-mouse IgG as the
secondary antibody. The flag-tagged PRLR shows expression at the plasma
membrane (a, nonpermeabilizing fixation condition) in the absence of
PRL, whereas it disappears from the plasma membrane in the presence of
PRL in similar fixation conditions (not shown); pronounced cytoplasmic
staining in all interphase cells is clearly seen, in endocytic vesicles
(b) and Golgi region in the presence of PRL, using permeabilizing
conditions. Obviously untransfected cells showed absence of labeling
(c). Magnification, x400.
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In addition, we analyzed by metabolic labeling the size of the PRLR
forms expressed and examined native BMGE cells for the presence of any
endogenous bovine PRLR. As shown in Fig. 3
, control cells (lane 1) and cells
transfected with pTM1 alone (lane 2) expressed no protein recognized by
the anti-PRLR U5 (17). However, when cells were transfected with either
pTM1-LPRLR (lane 3) or pTM1-SPRLR constructs (lane 4), proteins with
the correct molecular mass (95 kDa and 42 kDa, respectively) were
immunoprecipitated with U5 anti-PRLR antibody. Further efforts to
detect any endogenous bovine PRLR mRNA in nontransfected BMGE cells by
RT-PCR (Fig. 4
) or by Northern blot
(results not shown) were negative. Also, no detectable binding could be
obtained in BMGE cells, whereas cells transfected with LPRLR or SPRLR
constructs showed significant levels of receptors (Table 1
). Thus, BMGE cells appear to be a good
experimental system by which to study the functional activity of rat
PRLR isoforms.

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Figure 3. Isotopic Labeling of Short and Long PRLR Forms
Expressed in BMGE Transfected Cells
Cells were infected and transfected as described in Fig. 1 with
pTM1-LPRLR or pTM1-SPRLR constructs. They were labeled with
[35S]methionine for 90 min and lysed and the lysates were
subjected to immunoprecipitation with U5 monoclonal antibody. The
immunocomplexes were analyzed on 7.5% polyacrylamide-SDS gels. Lane 1,
normal BMGE cells; lane 2, cells transfected with pTM1 alone; lane 3,
cells transfected with pTM1-LPRLR; lane 4, cells transfected with
pTM1-SPRLR.
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Figure 4. RT-PCR Amplification of PRLR mRNA in BMGE Cells
A, Ethydium bromide staining of PCR products using primers 1 and 2. B,
Hybridization of the gel shown in panel A after transfer using an
internal probe. C, Ethydium bromide staining of PCR products using
primers of ubiquitous cyclophilin. Lane 1, Control 293 cells
transfected with LPRLR cDNA; lane 2, BMGE cells transfected with
LPRLRcDNA; lane 3, BMGE cells transfected with SPRLR cDNA; and lane 4,
nontransfected BMGE cells.
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PRL Signaling to the ß-Casein Gene Promoter in Transfected BMGE
Cells
BMGE cells were used to assay PRL signals to milk proteins by each
rat PRLR form. Because of the potential alterations produced on the
cellular environment by the vaccinia virus expression system, it was
advantageous to use a regular transient transfection system for this
purpose. Each PRLR construct was transiently transfected with a
ß-casein-luciferase construct by the calcium phosphate procedure and,
after the recovery period, the cells were treated, with or without
oPRL, for 24 h. The results obtained are shown in Fig. 5
. As expected, the PRL signal to the
ß-casein promoter was efficiently transduced by the LPRLR form and
resulted in an 8-fold induction in luciferase activity whereas the
SPRLR form had no activity.

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Figure 5. PRL Signaling to the ß-Casein Promoter in BMGE
Transfected Cells
Cells were transiently cotransfected as described in Materials
and Methods with the ß-galactosidase expression vector, the
ß-casein-luciferase construct, and the PRLR forms expression vectors
or empty expression plasmid as indicated. After a PRL stimulation (400
ng/ml) of 24 h, the cells were lysed, and the luciferase activity
was measured and normalized to the ß-galactosidase activity. Results
are expressed as fold of induction when compared with unstimulated
cells and represent the mean ± SEM of three
experiments.
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SPRLR Blocks PRL Signals to the ß-Casein Gene Promoter
In rat and mouse tissues, PRLR isoforms are expressed in different
proportions according to the hormonal environment associated with the
estrous cycle, pregnancy, and lactation (3, 4). We tried to reproduce
such in vivo situations by transiently transfecting BMGE
cells with a mixture of LPRLR and SPRLR constructs in varying
proportions (Fig. 6A
) or with different
amounts of the LPRLR construct alone as a control (Fig. 6B
). The amount
of transfected DNA was maintained at 6 µg by adding empty plasmid
when necessary. As can be observed in Fig. 6B
, the PRL signal was
efficiently transduced to the ß-casein gene promoter at all assayed
concentrations of LPRLR construct. Interestingly, cotransfection of the
LPRLR:SPRLR constructs in a 5:1 ratio, decreased by 35% the
transduction of the PRL signal to the milk protein gene promoter when
compared with the control (Fig. 6A
). This effect progressively
increased as a function of the increase in the SPRLR expression. When
the ratio of LPRLR to SPRLR was 1:5, PRL signaling to ß-casein gene
promoter was completely abolished. Previous experiments have shown that
increasing the ratio of SPRLR to LPRLR constructs in cotransfection
experiments was effectively related to an increased ratio of expressed
proteins (19). These results show a novel function of the SPRLR, which
we propose to be the silencing of the LPRLR signaling to milk protein
genes. The blocking effect of SPRLR was highly significant, even at the
lowest ratio of LPRLR to SPRLR assayed (5:1), which suggests that
PRLs effect on cells can be efficiently modulated by regulating the
expression of the PRLR isoforms.

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Figure 6. Effect of PRLR Short Form Coexpression with PRLR
Long Form on PRL Induction of ß-Casein Gene Promoter
BMGE cells were transiently cotransfected as described in
Materials and Methods with the ß-galactosidase
expression vector, the ß-casein-luciferase construct, and different
amounts of the PRLR isoform expression vectors. A, Cells were
cotransfected with different proportions of PRLR long and short form
expression vectors as indicated. B, Cells were cotransfected with
different amounts of the PRLR long form expression vector and the empty
expression vector (plasmid) as indicated. After a PRL stimulation (400
ng/ml) of 24 h, the cells were lysed, and the luciferase activity
was measured and normalized to the ß-galactosidase activity. Results
are expressed as fold of induction when compared with unstimulated
cells, and they represent the mean ± SEM of three
experiments.
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Previous studies have demonstrated that the distal tyrosine of the
LPRLR or Nb2PRLR is necessary for PRL activation of ß-casein gene
promoter (20). The mutant forms of the long and Nb2 PRLR, in which the
conserved distal tyrosines were mutated to phenylalanine, and which
were shown to have partially or totally reduced functional activity,
respectively, were used to test whether they could have a silencing
role in PRL signal to milk protein gene transcription when LPRLR and
these mutant forms were transiently cotransfected in cells. As shown in
Fig. 7A
, the mutant form of the PRLR in
which Tyr-580 was mutated to phenylalanine (Y580F) when cotransfected
with LPRLR decreased the transduction of PRL signal to ß-casein gene
promoter. Interestingly, the blocking effect detected by Y580F
expression was less pronounced than the one observed by SPRLR (Fig. 6A
). However, when these studies were extended to the mutant form of
the Nb2 PRLR in which the Tyr-382 was changed to phenylalanine (Y382F),
the block in LPRLR signal to ß-casein gene promoter was of about the
same magnitude as that detected for SPRLR (Fig. 7B
). The reduced
efficiency of Y580F to block PRL signal of the LPRLR can be attributed
to the fact that this mutant receptor retains a reduced functional
activity (3050%) whereas SPRLR or Nb2PRLR Y382F has no functional
activity (20). Thus, these data corroborate the essential role of the
distal tyrosine residue located at the C terminus of the LPRLR for
efficient PRL activation of ß-casein gene transcription and confirm a
silencing role of the SPRLR.

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Figure 7. Effect of PRLR Distal Tyrosine Mutants Coexpression
with PRLR Long Form on PRL Induction of ß-Casein Gene Promoter
BMGE cells were transiently cotransfected as described in
Materials and Methods with ß-galactosidase expression
vector, the ß-casein-luciferase construct, and different amounts of
PRLR long form and PRLR mutants. A, Cells were cotransfected with
different proportions of PRLR long form (WT) and Y580F mutant
expression vectors as indicated. B, Cells were cotransfected with
different proportions of PRLR long form (WT) and Y382F mutant
expression vectors as indicated. After a PRL stimulation (400 ng/ml) of
24 h, the cells were lysed, and luciferase activity was measured
and normalized to the ß-galactosidase activity. Results are expressed
as fold of induction when compared with unstimulated cells, and they
represent the mean ± SEM of three experiments.
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DISCUSSION
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In the present study we have established a system with which to
study a transcriptional signaling pathway for PRL in a more relevant
cell type than the one previously used, i.e a bovine mammary
epithelial cell line lacking endogenous PRLR. Our data demonstrate that
in these mammary gland-derived cells, the SPRLR has at least one
biological function in PRL signaling to milk protein synthesis: to
silence the PRL effect, supporting a novel view in the understanding of
PRL actions. These data are consistent with the evidence showing that
PRL or GH activates their receptors producing a 1:2 hormone-receptor
complex by a sequential dimerization (21, 22, 23, 24) and show that the
composition of PRL-PRLR complexes must be important for the effect of
PRL in the cells. Our results show that the PRL signal to the
ß-casein gene promoter is only mediated by the formation of
PRL-(LPRLR)2 complexes. This fact correlates well with
previously published evidence on PRL signaling in other cell types
(12, 13, 14, 15). The self-association of the intracellular domain of the LPRLR
forms allows proper docking of intracellular transducer molecules at
the proline-rich region (Box 1) and at the distal tyrosine of LPRLR
(15, 19). This distal tyrosine is essential for PRL signaling to the
milk protein genes because a LPRLR mutant for this residue results in a
decrease of signal transduction (19). This could imply that either the
specific amino acid sequence or the molecular space present between Box
1 and the distal tyrosine is necessary to anchor the transducer
molecule that binds to distal tyrosine, since Y580F expression did not
completely silence PRL signals as was true for SPRLR or Y382F.
Alternatively, the quaternary structure of the PRL-PRLR complex or the
associated transducer molecule may represent the structural basis for
the above mentioned differences. The transducer molecule that
associates with the distal tyrosine is unknown, but it has been
suggested that this phosphorylated tyrosine could be involved in
association with and activation of STAT5 (15, 19). When SPRLR was
coexpressed with LPRLR, PRL signals to the ß-casein gene promoter
were blocked in a SPRLR concentration-dependent manner. This fact
strongly suggests that PRL-(LPRLR-SPRLR) complexes are inactive,
which provides a rapid means of blocking PRL signals. In molecular
terms, there are two possible ways to explain the inability of
PRL-(LPRLR-SPRLR) complexes to transmit the PRL signal to the milk
protein gene promoters. The first is that the essential transducer
(STAT5) needs the two distal tyrosines provided by the self-association
of the LPRLR dimers to bind the complex, while the hybrid receptor
dimers have only one. The other is that this transducer could recognize
only one distal tyrosine that is externally exposed in
PRL-(LPRLR)2 complexes and intra- or intermolecularly
hidden in the PRL-(LPRLR-SPRLR) complexes. The expression of SPRLR
forms is therefore not meaningless. On the contrary, SPRLR expression
provides to cells an alternate tool to modulate PRL action. Similarly,
formation of homo- or heterodimers has been shown to modulate cell
signaling for TNF and Fas/APO1 (25). It remains to determine whether
PRL-(LPRLR-SPRLR) or PRL-(SPRLR)2 complexes are involved in
other functions of PRL. Using granulocyte macrophage colony stimulating
factor (GM-CSF) receptor/PRLR chimera, it has been shown recently that
Nb2PRLR-SPRLR heterodimers failed to induce proliferation in BA/F3
line, and this was related to an absence of activation of Jak2 and Fyn
whereas the GM-CSF/Nb2PRLR homodimers are fully capable of
GM-CSF-induced proliferation and activation of Jak2 and Fyn in the
BA/F3 transfectants (26). Taken together, these data indicate that the
SPRLR acts, through heterodimerization, as a dominant negative isoform
inhibiting both proliferation and transmission of a lactogenic signal
by the receptor complex. Such heterodimers have also been shown to
occur in human embryonic kidney fibroblast 293 cells after
cotransfection of SPRLR and LPRLR and result similarly in an absence of
activation of Jak2 (19).
New insights into the understanding of PRL effects can be derived from
two facts. The first is that PRLR isoforms are expressed in a
tissue-specific manner regulated by the hormonal situation associated
with the stages of the estrous cycle, pregnancy, and lactation (3, 4).
Second, the present data show that effects of PRL mediated by the
intracellular cascade used to express the milk proteins depend on the
composition of the PRLR dimers, and this concept could be extended to
other functions of PRL (26) and perhaps to other growth factor
receptors presenting isoforms with truncated intracellular domains
(27). In the mammary gland, the LPRLR is highly expressed both at the
end of pregnancy and during lactation. However, the ratio between SPRLR
and LPRLR increases at midlactation with respect to the end of
pregnancy. Thus, the formation of inactive PRLR heterodimers will
increase at midlactation, and the PRL effect mediated by the
intracellular signaling to ß-casein will decline. Thus, the SPRLR
form appears to play an active negative role in PRL signaling to milk
protein gene transcription.
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MATERIALS AND METHODS
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Plasmid Construction
Plasmid pTM1 was obtained from B. Moss (NIH, Bethesda, MD) (28).
The DNA sequence corresponding to the LPRLR and SPRLR were amplified by
PCR using the cDNAs cloned into the plasmid Bluescript SK (29, 30) as a
template, after 10 cycles of amplification. The 5'-oligonucleotide
primer for both cDNAs was
5'-CCGGCCGGCCATGGCATCTGCACTTGCTTT-3', and the downstream
3'-oligonucleotide primers were
5'-GGCCCCCCGGGCTATCAGTGAAAGGAGTGC-3' for LPRLR and
5'-GGCCCCCCGGGTTCAGTAGTCAAGTTCCCC-3' for SPRLR. The PCR
oligonucleotides generated an NcoI site in the first AUG and
a SmaI site in the 3'-untranslated sequence. The resulting
PCR products were digested with NcoI and SmaI and
cloned into the polylinker region of the expression vector pTM1
linearized with NcoI and SmaI.
Cell Culture and Transfection
BMGE cells (31) were grown in DMEM with 20% (vol/vol) FCS. For
transfection, cells were grown in dishes of M24 plates until they
reached 5060% of confluence. They were first infected with a
recombinant vaccinia virus encoding the T7 RNA polymerase (vT7) (32),
at a multiplicity of infection of 510 in 0.2 ml serum-free DMEM for
60 min at 37 C. The inoculum was then removed, and the cells were
washed twice with serum-free DMEM. Plasmids (0.5 µg/dish) were
transfected by a liposome-mediated method (33) using lipofectin (GIBCO
BRL, Gaithersburg, MD). Transfected cells were incubated for 1416 h
in serum-free DMEM before isotopic labeling and immunoprecipitation or
indirect immunofluorescence. For Scatchard analysis, functional
studies, and immunofluorescence studies with the anti-flag antibody,
cells were transfected by a calcium phosphate procedure (30).
Isotopic Labeling and Immunoprecipitation
Fourteen to 16 h after DNA transfection, BMGE cells were
washed twice with methionine-deficient DMEM and incubated in the same
medium containing 5 µCi L-[35S]methionine
(Amersham, Little Chalfont, U.K.) for 90 min at 37 C. Cells were washed
twice with PBS and lysed in 0.4 ml lysis buffer [10 mM
Tris-HCl, pH 7.4, 5 mM EDTA, 50 mM NaCl, 30
mM sodium pyrophosphate, 50 mM sodium fluoride,
1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonylfluoride, 1% (vol/vol) Triton X-100, 2 µg/ml
leupeptin, 5 µg/ml aprotinin, 1 µg/ml pepstatin A] for 30 min at 4
C. After centrifugation (15,000 x g, for 15 min at 4
C) to remove any insoluble material, the lysates were incubated with 2
µg U5 antibody (anti-PRLR monoclonal antibody) (17), 2.5 µg rabbit
anti-mouse Ig (RAM, Nordic Immunological Laboratoties, Tilburg, The
Netherlands), and 30 µl protein A-Sepharose (Sigma, St. Louis, MO)
for 4 h at 4 C. The immunocomplexes were washed three times with
lysis buffer and analyzed on 7.5% polyacrylamide-SDS gels. Gels were
dried and exposed to autoradiographic films (Eastman Kodak, Rochester,
NY).
Whole Cell Binding and Scatchard Analysis
Transfected cells were deprived of serum for an overnight
period. For Scatchard analysis, BMGE cells transfected with PRLR cDNAs
(4 µg/100-mm culture dish) were incubated with 100,000 cpm of
[125I]human (h)GH and increasing concentrations (050
pM) of unlabeled oPRL in 1 ml PBS containing 0.5% BSA.
Incubations were carried out at room temperature for 4 h. Cells
were washed twice with ice-cold PBS, solubilized in 1 ml 0.5
N, NaOH and counted in a counter. [125I]hGH
was prepared using chloramine T; specific activity ranged from 80140
µCi/µg.
RT-PCR and Southern Blot Analysis
cDNA synthesis and PCR amplifications were carried out as
described previously (34). For PCR amplification, we used a 25 mer
(primer 1) corresponding to nucleotides 77101 in the extracellular
domain and an antisense 23 mer (primer 2) corresponding to nucleotides
796818 in the membrane-proximal intracellular domain. Both primers
are common to the long (rat, cow) and short (rat) forms of the PRLR.
The amplification products were separated on 2% agarose gels, stained
with ethydium bromide, and transferred onto a Hybond N+ membrane
(Amersham). Southern hybridization with 5'-end-labeled oligonucleotide
probe was performed according to manufacturers instructions. To
control the efficiency of the reverse transcription, a control
amplification of the ubiquitous cyclophilin was performed on the same
sample as previously described (34).
Indirect Immunofluorescence
BMGE cells were grown onto coverslips and transfected as
described above. Fourteen to 16 h after transfection, they were
washed twice with PBS and fixed in cold methanol for 10 min at 4 C.
After extensive washing with PBS, coverslips were incubated for 2
h at room temperature in blocking buffer [PBS containing 3% (wt/vol)
BSA, 0.4% (vol/vol) Triton X-100], then incubated with anti-PRLR T1
monoclonal antibody (1:20 dilution) in PBS/3% (wt/vol) BSA for 2
h at room temperature. The cells were washed six times with PBS before
incubation for 1 h with rabbit anti-mouse fluorescein conjugate
(1:20 dilution) (17). Cells were observed with a Zeiss fluorescence
microscope (Carl Zeiss, Thornwood, NY), and photographs were taken
using Kodak 400 ASA film.
When using the anti-flag antibody, transfected cells were grown as
subconfluent monolayer cultures in Labtek chambers. For the
experiments, cells were harvested for 312 h in GC3 medium and
incubated with oPRL (400 ng/ml) for 10 min to 12 h; cells were
washed three times with PBS and fixed with 4% paraformaldehyde in 0.1
M phosphate buffer (pH 7.4) for 20 min or methanol (-20 C)
for 5 min. Samples were successively incubated with blocking solution,
mouse anti-flag monoclonal antibody (IBI Kodak; 10 µg IgG/ml)
overnight at 4 C, then washed and further incubated for 45 min with a
1:40 dilution of fluorescein isothiocyanate-conjugated goat anti-IgG.
Cells were then mounted in 50% glycerol in PBS and observed on a Zeiss
microscope.
Specificity control experiments included incubation of cells in the
absence of primary and/or secondary antibodies or with IgG contol
monoclonal antibodies. No immunofluorescence was detected in any of the
specificity control experiments.
Transient Transfection and Stimulation of the ß-Casein Gene
Promoter
BMGE cells were grown in a 60-mm culture dish in DMEM/20%
(vol/vol) FCS until they reached 50% confluence. They were starved
overnight in GC3 medium composed of 1:1 DMEM and Hams
F-12 medium supplemented with transferrin (10 µg/ml), insulin (80
mU/ml), glutamine (2.5 mM), and nonessential amino acids.
Cells were transfected by a calcium phosphate precipitation procedure
(30) with 3 µg pCH110 (ß-galactosidase expression vector from
Pharmacia, Uppsala, Sweden) and 1.5 µg plasmid ß-casein-luciferase
(carrying a fusion between the promoter region of the rat ß-casein
gene and the coding region of the luciferase gene) (20) and different
amounts of expression vectors for the PRLR forms: pER11
(pECE-PRLR short form), pER23 (pECE-PRLR long form) (29, 30), flag-tagged PRLR (19), pCMV-WT (pCMV-PRLR long form), pCMV-Y580F
(pCMV-PRLR long form mutant Y580F), and pCMV-Y382F (pCMV-PRLR Nb2 form
mutant Y382F) (20). After a glycerol shock, fresh GC3
medium was added and the cells were incubated in the presence or
absence of 18 nM oPRL (400 ng/ml) and dexamethasone (250
nM) for 24 h before being lysed. Luciferase activity
was measured in arbitrary light units and normalized to the
ß-galactosidase activity. Results of luciferase activity of
ß-casein promoter upon PRL stimulation (18 nM) are
expressed as fold induction compared with cells not stimulated with
PRL. Results represent the mean ± SEM of three
different experiments.
 |
ACKNOWLEDGMENTS
|
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We especially thank Oreste Gualillo for his help with the
transfections, Josefa González-Nicolás for her assistance
with the photography, and Dr. E. Martinez-Salas for a critical review
of the manuscript.
 |
FOOTNOTES
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Address requests for reprints to: Dr. Marc Edery, Institut National de la Santé et de la Recherche Médicale, Unité 344 Endocrinologie Moléculaire, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris, Cedex 15 France.
This work was supported by grants from Dirrección General de
Investigacion Científica y Técnica (PB930255) and the
Ramon Areces Foundation. The Universidad Autonoma de Madrid supplied
financial support to J.J. Berlanga for a short-term visit to the
Unité 344 of INSERM, Paris, France.
1 Current address: Departamento de Biologia Molecular-Centro de Biologia
Molecular Severo Ochoa, Universidad Autonoma de Madrid, 28049 Madrid,
Spain. 
Received for publication November 15, 1996.
Revision received June 9, 1997.
Accepted for publication June 17, 1997.
 |
REFERENCES
|
---|
-
Kelly PA, Djiane J, Postel-Vinay MC, Edery M 1991 The
prolactin/growth hormone receptor family. Endocr Rev 12:235251[Abstract]
-
Shirota M, Banville D, Ali S, Jolicoeur C, Boutin JM, Edery
M, Djiane J, Kelly PA 1990 Expression of two forms of prolactin
receptor in rat ovary and liver. Mol Endocrinol 4:11361143[Abstract]
-
Nagano M, Kelly PA 1994 Tissue distribution and regulation of
rat prolactin receptor gene expression. J Biol Chem 269:1333713345[Abstract/Free Full Text]
-
Clarke DL, Linzer DLH 1993 Changes in prolactin receptor
expression during pregnancy in the mouse ovary. Endocrinology 133:224232[Abstract]
-
Taniguchi T 1995 Cytokine signaling through nonreceptor
protein tyrosine kinases. Science 268:251255[Medline]
-
Rui H, Kirken RA, Farrar WL 1994 Activation of
receptor-associated tyrosine kinase JAK2 by prolactin. J Biol Chem 269:53645368[Abstract/Free Full Text]
-
Lebrun JJ, Ali S, Sofer L, Kelly PA 1994 Prolactin induced
proliferation of Nb2 cells involved tyrosine phosphorylation of the
prolactin receptor and its associated tyrosine kinase JAK2. J Biol
Chem 269:1402114026[Abstract/Free Full Text]
-
Dusanter-Fourt I, Muller O, Ziemiecki A, Mayeux P, Drucker B,
Djiane J, Wilks A, Harpur AG, Ficher S, Gisselbrecht S 1994 Identification of JAK protein tyrosine kinases as signaling molecules
for prolactin. EMBO J 13:25832591[Abstract]
-
Clevenger CV, Medaglia MV 1994 The protein tyrosine kinase
p59fyn is associated with prolactin (PRL) receptor and is
activated by PRL stimulation of T-lymphocytes. Mol Endocrinol 8:674681[Abstract]
-
Berlanga JJ, Fresno JAV, Martín-Pérez J,
García-Ruiz JP 1995 Prolactin receptor is associated with c-src
in rat liver. Mol Endocrinol 9:14611467[Abstract]
-
Ali S, Pellegrini I, Kelly PA 1991 A prolactin-dependent
immune cell line (Nb2) expresses a mutant form of prolactin receptor.
J Biol Chem 266:2011020117[Abstract/Free Full Text]
-
Lesueur L, Edery M, Ali S, Paly J, Kelly PA, Djiane J 1991 Comparison of long and short forms of the prolactin receptor on
prolactin-induced milk protein gene transcription. Proc Natl Acad Sci
USA 88:824828[Abstract]
-
ONeal KD, Yu-Lee L 1994 Differential signal transduction of
the short, Nb2, and long prolactin receptors. J Biol Chem 269:2607626082[Abstract/Free Full Text]
-
Das R, Vonderhaar BK 1995 Transduction of prolactins (PRL)
growth signal through both long and short forms of the PRL receptor.
Mol Endocrinol 9:17501759[Abstract]
-
Lebrun JJ, Ali S, Ullrich A, Kelly PA 1995 Proline-rich
sequence-mediated Jak2 association to the prolactin receptor is
required but not sufficient for signal transduction. J Biol Chem 270:1066410670[Abstract/Free Full Text]
-
Ali S, Chen Z, Lebrun JJ, Vogel W, Kharitonenkov A, Kelly PA,
Ullrich A 1996 PTP1D is a positive regulator of the prolactin signal
leading to beta-casein promoter activation. EMBO J 15:135142[Abstract]
-
Okamura H, Zachwieja J, Raguet S, Kelly PA 1989 Characterization and application of monoclonal antibodies to the
prolactin receptor. Endocrinology 124:24992508[Abstract]
-
Perrot-Applanat M, Gualillo O, Buteau H, Edery M, Kelly PA 1997 Internalization of prolactin receptor and prolactin in transfected
cells does not involve nuclear translocation. J Cell Sci 110:11231132[Abstract/Free Full Text]
-
Perrot-Applanat M, Gualillo O,Pezet A, Vincent V, Edery M,
Kelly PA 1997 Dominant negative and cooperative effects of mutant forms
of prolactin receptor. Mol Endocrinol 11:00000000
-
Lebrun JJ, Ali S, Goffin V, Ullrich A, Kelly PA 1995 A single
phosphotyrosine residue of the prolactin receptor is responsible for
activation of gene transcription. Proc Natl Acad Sci USA 92:40314035[Abstract/Free Full Text]
-
de Vos AM, Ultsch M, Kossiakof AA 1992 Human growth hormone
and extracellular domain of its receptor: crystal structure of the
complex. Science 255:257272
-
Fuh G, Colosi P, Wood WI, Wells JA 1993 Mechanism-based design
of prolactin receptor antagonists. J Biol Chem 268:53765381[Abstract/Free Full Text]
-
Hooper KP, Padmanabhan R, Ebner KE 1993 Expression of the
extracellular domain of the rat liver prolactin receptor and its
interaction with ovine prolactin. J Biol Chem 268:2234722352[Abstract/Free Full Text]
-
Rui H, Lebrun JJ, Kirken RA, Kelly PA, Farrar WL 1995 Jak2
activation and cell proliferation induced by antibody-mediated
prolactin receptor dimerization. Endocrinology 135:12991306[Abstract]
-
Boldin MP, Mett IL, Varfolomeev EE, Chumakov I, Shemer-Avni Y,
Camonis JH, Wallach D 1995 Self-association of the death domain of the
tumor necrosis factor (TNF) and Fas/APO1 prompts signaling for TNF and
Fas/APO1 effects. J Biol Chem 270:387391[Abstract/Free Full Text]
-
Chang WP, Clevenger CV 1996 Modulation of growth factor
receptor function by isoform heterodimerization. Proc Natl Acad Sci USA 93:59475952[Abstract/Free Full Text]
-
Ross RJM, Esposito N,Shen XY, Von Laue S, Chew SL, Dobson PRM,
Postel-Vinay M-C,Finidori J 1997 A short isoform of the human growth
hormone receptor functions as a dominant negative inhibitor of the
full-length receptor and generates large amounts of binding protein.
Mol Endocrinol 11:265273[Abstract/Free Full Text]
-
Moss B, Elroy-Stein O, Mijukami T, Alexander WA, Fuerst TR 1990 New mammalian expression vectors. Nature 348:9192[CrossRef][Medline]
-
Boutin JM, Jolicoeur C, Okamura H, Gagnon J, Edery M, Shirota
M, Banville D, Dusanter-Fourt I, Djiane J, Kelly PA 1988 Cloning and
expression of the rat prolactin receptor, a member of the growth
hormone/prolactin receptor gene family. Cell 53:6977[Medline]
-
Lesueur L, Edery M, Paly J, Clark J, Kelly PA, Djiane J 1990 Prolactin stimulates milk protein promoter in CHO cells cotransfected
with prolactin receptor cDNA. Mol Cell Endocrinol 71:R7R12
-
Schmid E, Schiller DL, Grund C, Stadler J, Franke WW 1981 Type-specific expression on intermediate filament proteins in a
cultured epithelial cell line from bovine mammary gland. J Cell Biol 96:3750[Abstract/Free Full Text]
-
Fuerst TR, Niles EG, Studier FW, Moss B 1986 Eucaryotic
transient-expression system based on recombinant vaccinia virus that
synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci USA 83:81228126[Abstract]
-
Felger PL, Gadaj TR, Holm M, Romau R, Chan HW, Wenz M,
Northrop JP, Ringold GM, Danielsen M 1987 Lipofection: highly
efficient, lipid-mediated DNA transfection procedure. Proc Natl Acad
Sci USA 84:74137417[Abstract]
-
Freemark M, Nagano M, Edery M, Kelly PA 1995 Prolactin
receptor gene expression in the fetal rat. J Endocrinol 144:285292[Abstract]