Dominant Negative and Cooperative Effects of Mutant Forms of Prolactin Receptor
Martine Perrot-Applanat,
Oreste Gualillo,
Alain Pezet,
Valérie Vincent,
Marc Edery and
Paul A. Kelly
INSERM Unité 344 Endocrinologie Moléculaire
Faculté de Médecine Necker 75730 Paris Cedex 15,
France
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ABSTRACT
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In addition to a long form of 591 amino acids
(aa), two other forms of PRL receptor (PRLR), differing in the length
of their cytoplasmic domains, have been identified in the rat. The Nb2
form, lacking 198 aa in the cytoplasmic domain, is able to transmit a
lactogenic signal similar to the long form, whereas the short form of
291 aa is inactive. The ability of PRL to activate the promoter of the
ß-casein gene or the lactogenic hormone responsive element fused to
the luciferase reporter was assessed in Chinese hamster ovary cells or
293 fibroblasts transiently transfected with PRLR cDNAs. The function
of the short form was examined after cotransfection of both the long
and short forms. These results clearly show that the short form acts as
a dominant negative inhibitor through the formation of inactive
heterodimers, resulting in an inhibition of Janus kinase 2 (JAK2)
activation. The present study also investigates the possible
participation of cytoplasmic receptors in the signal transduction
pathway, using cotransfection experiments and a new approach that
selectively determines the contribution of cytoplasmic receptors in the
process of signal transduction. We cotransfected Chinese hamster ovary
cells with two cDNA constructs: a cytoplasmic (soluble) form of the
receptor with a deleted signal peptide (
-19), which is unable to
bind PRL, and a functionally inactive receptor mutant (lacking box 1),
which is anchored in the plasma membrane and able to bind PRL. This
approach has allowed us to show that
-19, lacking expression at the
plasma membrane, can transduce the hormonal message, at least to a
limited extent (up to 30% of wild type efficiency), providing that
association/activation occurs with a PRL-PRLR complex initiated at the
cell surface level; box 1 of the cytoplasmic form is necessary to
rescue this partial transcriptional activity of the inactive mutant.
This partial recovery is also parallel to the partial activation of
JAK2, indicating that the signal transduction pathway implicated JAK2.
Our results provide evidence that heterodimerization of receptors can
be implicated either in the positive or in negative activation of gene
transcription.
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INTRODUCTION
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PRL is known to regulate a variety of physiological processes,
including reproduction, mammary cell growth and differentiation, and
immune functions (1), through its receptor (PRLR), which belongs to the
superfamily known as hematopoietin/cytokine/GH-PRL receptors (2). This
family includes the receptors for interleukins (IL-2 to IL-15),
granulocyte-colony stimulating factor, granulocyte macrophage colony
stimulating factor (GM-CSF), leukemia-inhibitory factor, ciliary
neurotrophic factor, erythropoietin, GH, PRL, thrombopoietin , and
leptin. After hormone binding, receptor dimerization, consisting of one
molecule of PRL and two molecules of receptor, appears to be the first
step by which PRL mediates its various actions (3). The activation of
the receptor, which does not possess an intrinsic tyrosine kinase,
leads to the rapid phosphorylation of an associated cytoplasmic
tyrosine kinase, Janus kinase 2 (JAK2), which is necessary for the
phosphorylation of the receptor and signal transduction molecules (for
review, see Refs. 4, 5, 6); box 1 (a proline-rich motif) and the adjacent
residues upstream of box 2 in the cytoplasmic proximal region (near the
membrane-spanning domain) of cytokine receptors are sufficient for JAK
kinase association/activation (7, 8). The major downstream targets of
PRL-activated JAK kinases include members of the Shc-Ras pathway (9)
and some members of the signal transducers and activators of
transcription (STAT) protein family (10), including STAT1, STAT3, and
STAT5 or MGF (mammary gland factor) (11, 12). Members of the STAT
family that possess a SH2 domain require tyrosine phosphorylation for
activation. In addition to a long form of 591 amino acids (aa), two
other forms of PRLR differing in the length of their cytoplasmic
domains have been identified in the rat. As is true for the long form,
the Nb2 form, lacking 198 aa in the cytoplasmic domain, is able to
transmit the lactogenic signal, while a short form of 291 aa is
inactive in lactogenic bioassays (13, 14, 15, 16). In the first part of this
study, we investigated the effect of the short form of rat PRLR on the
activity of the long form of PRLR in differentiated cells.
Signal transduction through cytokine receptors, as for receptor
tyrosine kinases, is believed to occur almost exclusively at the level
of the plasma membrane. Several growth factors [epidermal growth
factor (EGF), insulin], as well as PRL, induce the rapid
internalization of their receptors by hormonally responsive cells using
a receptor-mediated process (17, 18, 19, 20, 21). PRL and PRLR endocytosis and
recycling remain poorly understood, as compared with other membrane
receptors (such as insulin, EGF, low-density lipoprotein, transferrin;
see Refs. 22, 23 for a review). Rapid internalization of growth
factors [EGF, platelet-derived growth factor (PDGF)] and their
receptors, which belong to the receptor tyrosine kinase family, into
endosomes is traditionally thought to attenuate the ligand-induced
response; however, there is also growing evidence for selective and
regulated signal transduction occurring within the endosome, based on
the presence of an active growth factor tyrosine kinase and
tyrosine-phosphorylated signaling molecules. In fact, there is a
paucity of data to associate receptors and signaling pathways to
membrane trafficking, including the first step, endocytosis. For
several growth factors (EGF, PDGF), both the mechanism of endocytosis
and signal transduction require the phosphorylation of tyrosine
residues by the kinase domain located in the cytoplasmic region of the
receptor (22, 23). Internalization and signal transduction by the GH
receptor are mediated by different regions of the cytoplasmic domain
(24). Similar studies concerning internalization of the PRLR are
currently in progress, and for other cytokine receptors, the
requirement of a tyrosine kinase being directly required for
internalization remains unknown. In the second part of this study, we
have investigated the possible implication of cytoplasmic receptor
constructs in signal transduction.
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RESULTS
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PRLR Short Form Is a Dominant Negative Inhibitor of the Long Form
of Receptor
The different forms of the rat PRLR, differing in the length of
their cytoplasmic domains (13), were efficiently expressed when
transfected in COS-7 cells, as judged by their PRL-binding capacity and
affinity and immunolocalization using specific anti-PRLR antibodies
(Table 1
). To analyze the influence of the expression of the short form
of PRLR upon transcriptional activity of the long form, we used a
functional transcription assay, which consists of cotransfecting a
ß-casein/luciferase reporter gene construct with PRLR cDNA (long
form, short form, or mixture of long and short forms). Initial studies
were performed in Chinese hamster ovary (CHO) cells, which have no
detectable endogenous PRLR and are thus a suitable model system for
these studies. As shown in Fig. 1A
, the long form of
PRLR, in contrast to the short form, activated the
ß-casein/luciferase reporter gene upon PRL stimulation. This
functional activation of the long form of PRLR was reduced in the
presence of the short form of PRLR. The ß-casein/luciferase induction
progressively decreased in the presence of increasing amounts of the
short form of PRL-R. This competition of the short form PRLR with the
activity of the long form was observed at several concentrations of
ovine (o)PRL (400 ng to 10 µg/ml) (not shown). Similar results have
been obtained with a bovine mammary epithelial cell line (BGME)
cotransfected with long and short forms of rat PRLR cDNA (not
shown).

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Figure 1. Inhibition of the Long Form of PRLR Activity by the
Short Form Receptor
A, CHO cells were cotransfected with a construct that contains a
milk protein (ß-casein) gene promoter fused to the reporter gene of
the firefly luciferase (plasmid-casein/luciferase) and PRLR cDNA, as
described in Materials and Methods. After incubation
with oPRL (400 ng/ml) and dexamethasone (250 nM) for
48 h at 37C, cells were assayed for luciferase activity.
Cotransfections experiments included the transfection of either the
long form or the short form of PRLR cDNA alone (long 6:0 or short 0:6,
respectively), or a mixture of both the long and short forms of PRLR
cDNAs. Control long or short form/luciferase-transfected cells had a
basal luciferase activity (without hormone) of 500 RLU/U ßgal. In
these experiments, results of luciferase activity were assayed in cells
transfected with several ratios (6:0 to 0:6, for 6 µg total cDNA) of
long and short forms of PRLR cDNA. Results of luciferase activity of
ß-casein gene promoter upon PRL stimulation (400 ng/ml) are expressed
as fold-induction compared with cells not stimulated with PRL. Results
represents the means ± SEM of three to five
independent experiments. B, Same experiments in 293 cells using LHRE
fused to the reporter gene of firefly luciferase. Results of luciferase
activity were assayed in cells transfected with several ratios (1:0 to
1:5, for 6 µg total cDNA) of long and short forms of PRLR cDNA.
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To study in detail the dominant negative action of the short form,
further studies were conducted in the human embryonic kidney fibroblast
293 cell line in which both functional assay and Western blot analysis
are possible due to the high level of expression of plasmids under the
control of the CMV promoter in this cell line. As shown in Fig. 1B
, in
cotransfection experiments, increasing concentration of the short form
of PRLR resulted in a stepwise decrease in the functional activity of
the long form of PRLR. Luciferase fold induction using the ß-casein
promoter was routinely in the range of 57, whereas it was much higher
using the lactogenic hormone response element (LHRE) construct (see
Materials and Methods) for which we obtained maximal
activation of 18-fold for the long form alone. Again, a drastic
inhibition (90%) was obtained with a 1:5 ratio of long to short form
of PRLR cDNA, similar to what was obtained in CHO (Fig. 1
) or BMGE
cells, (J. J. Berlanga, J. P. Garcia-Ruiz, M. Perrot-Applanat, P. A.
Kelly, and M. Edery, manuscript submitted). The same results were
obtained when the intermediate Nb2 form was used instead of the long
form of the PRLR (data not shown).
Mechanisms of the Dominant Negative Action of the Short-Form
PRLR
To investigate how this dominant negative action of the short form
occurs, two series of experiments were conducted. In the first set of
experiments, we analyzed by Western blots the activation of JAK2, which
is known as the primary kinase activated during PRL signaling.
In 293 cells cotransfected with long and short forms of the PRLR cDNA,
there was a drastic reduction in the activation of JAK2 upon PRL
stimulation, as shown by Western blot revealed with antiphosphotyrosine
antibodies (Fig. 2A
, lanes 34), whereas cells
transfected with the long form only displayed a clear activation of
JAK2 upon PRL stimulation (Fig. 2A
, lanes 1 and 2). Similar results
were obtained with cotransfection of Nb2 and short forms of PRLR cDNA
(Fig. 2A
, lanes 58). Control experiments in which blots were revealed
using JAK2 antibody indicate a similar level of JAK2 expression in
cells stimulated or not by PRL (see Fig. 2B
). As has been previously
described (8), more JAK2 was associated with the Nb2 form of PRLR (Fig. 2A
, lanes 5 and 7). As is common in transient transfections, some
slight differences in the amount of JAK2 expressed was observed (Fig. 2A
, lanes 3, 5, and 7). Also, the level of tyrosine phosphorylation of
PRLR was greatly reduced when the short and long forms of PRLR were
cotransfected (data not shown). Thus, the short form of the rat PRLR
exerts a dominant negative effect on the activation of the long-form
receptor, via the inhibition of the activation of JAK2 kinase.

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Figure 2. Inhibition of the Activation of JAK2 by the Short
Form of PRLR
293 cells (2.5 x 106/lane) were cotransfected with 1
µg cDNA encoding the long form of PRLR alone (control, lanes 12) or
together with 5 µg cDNA encoding the short form of PRLR (lanes 34);
with 1 µg cDNA encoding the Nb2 form of PRLR alone (lanes 56); or
together with 5 µg cDNA encoding the short form of PRLR (lanes 78);
and with 0.5 µg of cDNA encoding JAK2. After stimulation (+) or not
(-), cell lysates were processed as described in Materials and
Methods and immunoprecipitated with an anti-JAK2 monoclonal
antibody. Immunoprecipitates were resolved on 7% SDS-PAGE, and the
blots were probed with antiphosphotyrosine antibody (panel A) or
anti-JAK2 antibody (panel B).
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In the second set of experiments, we investigated whether the dominant
negative effect of the short form resulted from the formation of
heterodimers between long and short forms of PRLR. For this purpose, we
generated a cDNA of the long form of the rat PRLR containing an
oligonucleotide encoding a Flag epitope inserted just before the
N-terminal amino-acid (named Flag-tagged PRLR); the encoded protein is
specifically recognized by anti-Flag antibodies (25, 26). Complementary
DNA encoding Flag-tagged PRLR long form was cotransfected with the
nontagged short form cDNA in 293 cells, proteins were
immunoprecipitated using anti-Flag antibody and further analyzed by
Western blot using either anti-Flag antibody or anti-PRLR (U5). When
blots were analyzed with anti-Flag antibody, a single band appeared in
cells cotransfected with both PRLR cDNAs and in control cells
transfected with PRLR long form alone; this band correspond to the
Flag-PRLR (95 kDa) (Fig. 3
, lanes 14). On the other
hand, when monoclonal anti-PRLR antibody (U5) was used for blotting,
two bands of 95 kDa and 42 kDa were revealed in cotransfected cells
upon PRL stimulation (Fig. 3
). In control cells transfected with
Flag-PRLR alone or in control cells cotransfected in the absence of
PRL, only one band (95 kDa) was observed. The two bands present in
cotransfected cells upon PRL stimulation correspond to the long and
short forms of PRLR, indicating that the Flag antibody, which is
specific for the long form of PRLR, was able to coimmunoprecipitate
long and short forms of PRLR resulting from heterodimerization.

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Figure 3. Heterodimerization of Short and Long Forms of PRLR
293 cells were cotransfected with 1 µg cDNA encoding a Flag-tagged
long form of PRLR(L) alone (lanes 12 and 56) or together with 5
µg cDNA encoding the short form of PRLR(S) (lanes 34 and 78).
Cell lysates were processed as described in Materials and
Methods and immunoprecipitated with an anti-Flag monoclonal
antibody. Immunoprecipitates were resolved on 8% SDS-PAGE, and the
blots were probed with an anti-Flag monoclonal antibody (lanes 14) or
an anti-PRLR monoclonal antibody (U5) (lanes 58).
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Synthesis of Inactive Mutants of the PRLR Expressed at the
Cell Surface
We constructed mutants of PRLR (Fig. 4A
), which are inactive in the
ß-casein/luciferase reporter assay when expressed in CHO cells, but
retain the characteristics of the wild type receptors with respect to
their expression at the cell surface, binding affinity, and endocytosis
(Table 1
). A mutant of the Nb2 form of PRLR in which the four prolines
in box 1 are replaced by alanine (4P/A) has no activity due to its lack
of association and activation of JAK2 (8). As shown in Fig. 4B
and
Table 1
, mutations in the box 1 region in the PRLR long
form (4P/A and P250L, see Materials and Methods) resulted in
the loss of ability to transduce the PRL signal, but neither binding
nor ability to internalize of the mutants was altered (see Fig. 4C
).
Also, the level of expression of these mutants was similar to that of
the wild type receptor, as evaluated using Western blots (Fig. 4D
).

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Figure 4. Functional Expression of Wild Type and Mutants
PRLRs
A, Schematic representation of wild type and mutant PRLR: long form,
P250L (substitution of proline 250 to leucine), 4P/A (substitution of
prolines 245, 246, 248 and 250 to alanine), -19 (deletion of the
signal peptide), -19, 4P/A (deletion of the signal peptide and
substitution of prolines 245, 246, 248, and 250 to alanine), NLS
(addition of a nuclear localization signal), short form, Nb2 form and
Nb2-T322 (truncation of the cytoplasmic tail of the Nb2 form).
Generation of the mutants via a recombinant PCR strategy is described
in Materials and Methods. B, Stimulation of functional
activity by PRL. Cells were cotransfected with ß-casein/luciferase
construct and PRLR cDNAs and assayed for luciferase activity after
stimulation with (or without) oPRL (400 ng/ml, 48 h) as described
in Materials and Methods. Results are expressed as
fold-induction of luciferase activity in the presence of PRL, as
compared with the activity in the absence of the hormone. Mutation of
box 1 or deletion of the signal peptide of PRLR leads to inactive
PRLRs. Values represent means ± SEM of five
independent experiments. C, Receptor-mediated endocytosis of wild type
and mutants PRLR in COS-7 cells. Cells were transfected with PRLR cDNA
wild type (long), Nb2, 4P/A, or -19 + 4P/A mutant form of the PRLR and incubated
with [125I]hGH. The kinetics of internalization were
determined after different periods of incubation, as described in
Materials and Methods. Kinetics of internalization of
P250L mutant is similar to that of 4P/A mutant. These results are
representative of three independent experiments performed in duplicate.
D, Expression of different mutant forms of the PRLR. 293 cells were
transfected with 2 µg cDNA encoding the PRLR wild type (WT), the
P250L, the 4P/A, and the -19 mutant forms of the PRLR. Cell lysates
were processed as described in Materials and Methods and
immunoprecipitated with a monoclonal antibody (U5) directed against the
extracellular domain of the PRLR, except for the -19, which was
immunoprecipitated with an anti-Flag monoclonal antibody M2 ( Flag).
Immunoprecipitates were separated using an 8% SDS-PAGE, blotted onto
PVDF membranes and then probed with an anti-PRLR antibody (U5) for WT,
P250L, and 4P/A or an anti-Flag antibody for -19.
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Synthesis of Inactive PRLR Mutants Expressed in the Cytoplasm
(Soluble Mutants)
We next constructed a cytoplasmic (soluble) form of the PRLR, by
deleting the sequence encoding the signal peptide (
-19) (Fig. 4A
)
(see Materials and Methods), which resulted in a mutant
receptor unable to enter the rough endoplasmic reticulum during the
biosynthetic pathway and thus, to be targeted to the plasma membrane.
Immunofluorescence analysis confirmed its cytoplasmic localization,
with a diffuse pattern clearly different from the immunofluorescence
pattern corresponding to the normal protein secretory pathway (RER,
Golgi apparatus, and vesicles as shown in Fig. 5
and in
Ref. 25). This mutant receptor was functionally inactive, as judged by
the absence of PRL binding and ß-casein/luciferase transcriptional
activity in the presence of PRL (Fig. 4B
). Also, the receptor lacking
the signal peptide was equally stable compared with the wild type
receptor, as observed by Western blot (a single band of 85 kDa), and
immunofluorescence after 012 h PRL stimulation (Fig. 4D
and Fig. 5
).
Characteristics of the various mutant PRLRs are summarized in Table 1
.

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Figure 5. Immunofluorescence Localization of PRLR or -19
Mutant Receptor
COS-7 cells were transfected with cDNAs encoding PRLR (a, b, and c) or
-19 mutant cDNA (d) and processed for indirect immunofluorescence,
as described in Materials and Methods. PRLR was
expressed at the cell surface (c, as observed in non permeabilized
cells) and in membranous compartments (probably rough endoplasmic
reticulum, Golgi area, and vesicles as observed in permeabilized cells;
see the Golgi area and vesicles in panel a); in contrast, -19 mutant
receptor is expressed within the cytoplasm as a soluble protein (d), as
deduced from the diffuse pattern characteristic of cytoplasmic protein
(50 ). b, Phase contrast. Magnification, x300. The expression of the
-19 mutant receptor is stable over a 12-h stimulation
(25 ).
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Soluble
-19 PRLR Mutant Rescues Membrane-Inactive Box 1 PRLR
Mutant
When CHO cells were cotransfected with a PRLR cDNA encoding a box
1 inactive mutant (4P/A or P250L) and a cDNA encoding a cytoplasmic
form of the receptor (
-19) along with the ß-casein/luciferase
reporter gene, transcriptional activity was significantly
(P < 0.05) recovered upon PRL stimulation (400 ng/ml)
up to 28 ± 3% of wild type efficiency (Fig. 6
),
suggesting a cooperative effect of the two mutant forms of receptor.
This increase was PRL-dependent inasmuch as basal luciferase activity
of the two cotransfected mutants was not modified in the absence of
hormone, as compared with basal activity of each mutant alone. Similar
results were observed at various concentrations of oPRL (400 ng/ml to
10 µg/ml) (not shown). Maximal recovery was obtained with similar
microgram amounts of cotransfected cDNAs (3:3 to 4:2 ratios of cDNAs of
cytoplasmic vs. plasma membrane mutants). These results
suggest that some functional hormone-dependent interaction may occur
between an inactive membrane-bound PRLR and a cytoplasmic mutant.
As the box 1 region is known to be necessary for signal transduction
(8, 27), we investigated the possible involvement of box 1 of the
cytoplasmic mutant on the partial recovery of transcriptional activity.
Cotransfection experiments using PRLR cDNAs encoding an inactive cell
surface receptor and a cytoplasmic mutant, both receptors being mutated
in box 1 (mutants 4P/A or P250L and
-19, 4P/A, see Fig. 4
, A and B),
failed to activate the ß-casein/luciferase reporter gene (Fig. 6
).
This suggests that an intact box 1 of the cytoplasmic form of the
receptor is necessary for the recovery of partial transcriptional
activity, after PRL stimulation.
In contrast, no functional activity could be recovered when the
inactive short form of PRLR or the truncated mutant T322 of the Nb2
form (see Fig. 4B
) was cotransfected with the
-19 (Fig. 7
), although these forms are binding forms and are able
to internalize (Table 1
). These results probably reflect the
requirement for distal C-terminal sequences in the PRLR cytoplasmic
domain (in addition to the box 1 region) for the partial recovery of
transcriptional activity. Association of the two receptor forms
probably occurs in the cytoplasm because cotransfection experiments
with a nuclear mutant, generated by addition of a nuclear localization
sequence (NLS) in the (
-19) PRLR mutant (see Fig. 4A
), and an
inactive box 1-mutated PRLR did not permit recovery of transcriptional
activity of the inactive mutant (long form) expressed at the plasma
membrane (Fig. 7
). This further confirms that the partial recovery of
transcriptional activity of the two inactive mutants is only observed
for receptor interactions occurring in the cytoplasm and in the
presence of PRL.

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Figure 7. Inactive Short and Mutant Nb2 Receptors Are Unable
to Be Rescued by a Cytoplasmic PRLR Mutant
Cells were transfected with ß-casein/luciferase construct and cDNAs
of the short or mutant Nb2 T322 and -19. Control experiments
included transfection with cDNAs encoding the long form, -19, Nb2
T322, or NLS+4P/A. Values represent the means ± SEM
of three independent experiments performed in duplicate.
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To determine whether the partial activation of transcription observed
after cotransfection of
-19 and 4P/A resulted from a partial
activation of the JAK/STAT pathway, the activation of JAK2 was
determined as described before in 293 cells (8). Briefly, 293 cells
were cotransfected with
-19, and 4P/A and JAK2 cDNAs,
immunoprecipitated with anti-PRLR monoclonal antibody, and further
analyzed by Western blot using antiphosphotyrosine antibody. Whereas
the transfection of the long form of PRLR used as control resulted, as
previously described (8), in the phosphorylation of JAK2 (130 kDa) and
PRLR (95 kDa), the cotransfection of
-19 and 4P/A resulted in a
reduced, albeit still significant, phosphorylation of JAK2 and PRLR
after PRL stimulation (Fig. 8
).

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Figure 8. Rescue of an Inactive PRLR (Long Form) by a
Cytoplasmic PRLR Mutant involves JAK2 Activation
293 cells were transfected with 2 µg cDNA encoding the long form of
the PRLR (lanes 12) or with cDNAs encoding the 4P/A and -19 mutant
forms (lanes 34), along with 0.5 µg cDNA encoding JAK2. After
stimulation (+) or not (minus]) with 400 ng/ml oPRL (18
nM), cell lysates were immunoprecipitated with a monoclonal
antibody (U5) against the PRLR. Immunoprecipitates were separated using
a 8% SDS-PAGE, blotted onto PVDF membranes, and then probed with an
antiphosphostyrosine antibody. The positions of JAK2 (130 kDa) and PRLR
(95 kDa) are indicated on the ight.
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DISCUSSION
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Since the cloning of PRLR cDNAs revealed their existence in
multiple forms (i.e. long, short, and intermediate) (13),
extensive studies on the function of each form have been reported.
Using CHO cells cotransfected with PRLR cDNA and a reporter gene (CAT
or luciferase) under the control of a milk protein gene promoter, it
has previously been shown that both the long and intermediate forms of
the rat PRLR were able to induce expression of the reporter gene
(14, 15, 16), whereas the short form was inactive (14). On the other hand,
both the long and short forms of the PRLR were shown to transmit a
mitogenic signal in transfected NIH 3T3 cells (15). Using
cotransfection experiments in CHO and 293 cells, the present study
demonstrates one additional function of the short form of PRLR in PRL
signaling of milk protein gene activation: that it can act as a
negative regulator, supporting a novel view in the understanding of PRL
action. Since the first step in PRL action is the dimerization of the
receptor induced by PRL (3), the composition of PRLR-PRL complexes is
fundamental to the ability of PRL to stimulate milk protein gene
transcription. The homodimerization of PRLR allows proper docking of
transducing molecules both at the proline-rich region (box 1), such as
JAK2 (8), and at a C-terminal tyrosine (28). This tyrosine has been
shown to be responsible for activation of gene transcription in the Nb2
PRLR and to be the major regulator in the long form of PRLR (28). It
has been suggested that this phosphorylated tyrosine could be involved
in association and activation of STAT5 (12); the region surrounding
this residue may contain an interaction site for the tyrosine
phosphatase PTP1D (29).
When the short form is coexpressed with the long form of the PRLR, PRL
signaling to milk protein genes is blocked in a dose-dependent manner.
This inhibitory activity of the short form of PRLR could reflect the
sequestration of PRL by an increase of inactive homodimers of short
form or that heterodimerization of long and short forms of PRLR
prevents PRL signaling (nonfunctional heterodimers). The dominant
negative effect of the short form is further supported by the fact that
JAK2 activation, as well as PRLR phosphorylation, which represent the
earliest events in PRLR activation after PRL binding, were inhibited
after cotransfection experiments of long and short forms. Evidence for
the formation of heterodimers between long and short forms of PRLR is
given from cotransfection experiments using a Flag-tagged long form PRL
cDNA and a nontagged short-form PRLR cDNA (Fig. 3
). This dominant
negative effect of the short form implies that the STAT protein needs a
pair of distal receptor domains, presumably the two distal
phosphotyrosines, for proper docking, and that homodimerization of
receptors is required for proper signaling. Further experiments
involving specific phenylalanine mutations are required to correlate
receptor phosphorylation and biological activity. Finally, the ratio of
expression of the short to the long form of PRLR has been shown to vary
in a tissue-specific manner and also as a function of physiological
state, providing cells a powerful means of modulating PRL action. A
clear example is illustrated by the mammary gland and liver, in which
reverse ratios of long and short forms exist (30). A similar mechanism
of modulation of cell signaling through formation of homo- and
heterodimers has been seen for insulin, erythropoietin, TNF
, and
Fas/APO1 effects (31, 32, 33). However, whether PRL-(Long PRLR-Short PRLR)
or PRL-(Short PRLR)2 complexes are involved in other
functions of PRL is still an open question. It is interesting to note
that, using GM-CSF receptor/PRLR chimera, it has been shown that
Nb2PRLR-Short PRLR heterodimers failed to induce proliferation in BAF3
line, and this was related to an absence of activation of JAK2 and Fyn,
whereas the GM-CSF receptor/Nb2PRLR homodimers are fully capable of
GM-CSF-induced proliferation and activation of JAK2 and Fyn in the BAF3
transfectants (34). Together, these data and the present findings
indicate that the Short PRLR functions as a dominant negative isoform,
inhibiting both proliferation and activation of milk protein gene
transcription by the receptor complex through heterodimerization (Fig. 9
). Indeed, such heterodimers are shown to occur in
human embryonic kidney fibroblast 293 cells.

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Figure 9. Hypothetical Model of Inactive and Active PRLR
Heterodimers
A, Binding of PRL to its receptor induces heterodimerization. JAK2 is
associated to the PRLR through the box 1 sequence (black
box) in the cytoplasmic domain, but there is no activation of
JAK2 or of intracellular PRLR tyrosines. B, Binding of PRL to its
receptor induces PRLR dimerization. Recruitment of cytoplasmic box 1
active PRLR results in transphosphorylation (dashed
arrow) of JAK2 itself as well as the PRLR intracellular
tyrosines that provide docking sites for Stat5 association and
activation leading to signal transduction.
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We also investigated the possible implication of cytoplasmic receptors
in signal transduction of PRLR, as such soluble receptors have been
reported (35). For this purpose, we developed a new approach that
selectively determines the contribution of cytoplasmic receptor
constructs in the process of signal transduction. We cotransfected CHO
cells with two cDNA constructs:
-19 and 4P/A or P250L, the last two
encoding receptors anchored in the plasma membrane and able to bind
PRL. The
-19 receptor is not processed through the endoplasmic
reticulum and the Golgi apparatus where the formation of disulfide
bonds and glycosylation occurs and does not bind PRL even when a
cytosolic preparation is used (Table 1
), probably due to an improperly
folded extracellular domain. This approach has allowed us to show, for
the first time, that a mutant of the PRLR, lacking expression at the
cell surface can transduce the hormonal message, at least to a limited
extent (up to 30% of wild type efficiency), providing cooperation via
association/activation occurs with a PRL-PRLR complex initiated at the
cell surface. The rescue and activation of a binding deficient insulin
receptor has been previously reported by a membrane-adjacent receptor
(36). That only partial recovery of transcriptional activity was
observed in our system can be explained by the fact that we used a
soluble receptor designed only to detect a potential cytoplasmic
activation pathway. Assuming that 100% functional activity corresponds
to the combination of plasma membrane and cytoplasmic contributions,
30% recovery of activity observed in this study could represent the
maximal contribution of intracellular receptors or could be reflective
of the maximal activation possible when only a subset of
-19, that
portion which is in the membrane-proximal region of the cytoplasm, is
available to associate with the box 1 mutant receptor. In addition,
partial recovery also could be due to the stoichiometry involving
-19 and the inactive membrane-bound mutants, which might limit the
potential formation of tetramers and oligomers.
The fact that box 1 of
-19 is necessary for the partial
transcriptional activity of the 4P/A mutant expressed at the plasma
membrane suggests that the biological responses observed in this study
probably occur through box 1, as is true for wild type and Nb2 PRLRs
(4, 5, 28). This partial recovery is also parallel to the partial
activation of JAK2 and phosphorylation of the PRLR after cotransfection
and PRL stimulation, indicating that the signal transduction pathway
implicates JAK2 and box 1 of PRLR. The lack of recovery of milk protein
gene transcription with Nb2 T322 or the short form suggests that other
regions of the cytoplasmic domain, in addition to box 1, are involved
in activation. Indeed, cytoplasmic segments of PRLR have been shown to
provide cooperating or conflicting intracellular signals (12). Such a
negative domain has been shown for the erythropoietin receptor, which
contains a site of interaction for the tyrosine phosphatase PTP1C or
SHP1 (37). Also, the fact that box 1 of
-19 and the full-length
cytoplasmic tail of 4P/A or P250L mutant must both be present for
partial recovery of transcriptional activity support the concept of
heterodimerization and transphosphorylation of PRLRs, as has also been
described for the insulin receptor (38) (Fig. 9
). Partial activation
was obtained using an inactive box 1 PRLR mutant for which endocytosis
after PRL stimulation is not altered. That PRL interacts with its
receptor intracellularly under normal conditions has been previously
described in the 2351 mammotroph PRL-secreting cell line (39);
furthermore, cytosolic PRLRs have been identified (35); and PDGF
receptor activation in intracellular compartments by an autocrine
mechanism has been described in v-sis-transformed cells
(40). Further experiments using mutagenesis of the soluble form of the
PRLR are needed to determine the interaction domain(s) in the
cytoplasmic tail of the two receptors.
This study also provides evidence that box 1, a proline-rich region, is
not required for internalization of the PRL-PRLR complex. The fact that
box 1 is required for signal transduction, but not for endocytosis,
contrasts with the situation observed for several growth factor
receptors, including those for EGF and PDGF, for which phosphorylation
of tyrosine residues by the kinase domain is required (22). Recently,
it has been shown that a mutant EGF receptor lacking a functional
tyrosine kinase cannot undergo efficient recruitment into coated pits
(41). However, it is not impossible that a PRLR-associated kinase,
distinct from JAK2, could be required for the PRLR internalization. In
addition, the domains required for internalization and signal
transduction may be located in different regions of the cytoplasmic
domain of the PRLR.
Our experiments, based on a new approach that selectively determines
the contribution of cytoplasmic receptor, suggest that the association
of the two inactive mutants can occur at the cytoplasmic face of the
cell membrane, or anywhere in the cytoplasm where 1) the soluble form
is present in high amounts, 2) the membrane form is endocytosed, and 3)
all necessary signal-transducing molecules are present and can
associate with such receptor. In the latter case, the process of signal
transduction generated by an activated PRLR at the cell surface can be
prolonged inside the cell through heterodimerization. However,
experiments using a PRLR box 1 mutant, which also cannot be
endocytosed, are necessary before conclusions can be made about whether
the partial rescue of transactivation by the cytoplasmic PRLR rely on
endocytosis. Although signal transduction through cytokine receptors,
as is true for tyrosine kinase receptors, is believed to occur almost
exclusively at the level of the plasma membrane, a model in which
endocytosis plays a role in transmitting a ligand-dependent signal has
been proposed on theoretical grounds (22, 23, 42): 1) rapid
internalization of the growth factor and its receptor, and 2) presence
of an active growth factor tyrosine kinase and tyrosine-phosphorylated
signaling molecules within the endosome.
Thus, heterodimerization of the PRLR can result either in an inhibition
or an activation of gene transcription, depending of the formation of
active or inactive dimers, which presents an alternative in modulating
PRL action. A model that depicts these two mechanisms is shown in Fig. 9
.
 |
MATERIALS AND METHODS
|
---|
PRLR and Mutants
The full-length cDNA for the long, short, and Nb2 forms (16, 43)
was used for these studies. Several mutants of the PRLR were generated
from the long form and the Nb2 form of the rat PRLR via a recombinant
PCR strategy, as follows.
The construction of P250L and 4P/A mutants was generated in the pR/CMV
vector (Invitrogen, San Diego, CA) as a template, by substitution of
proline 250 to leucine and prolines 245, 246, 248, and 250 to alanine,
respectively.
Deletion of the signal peptide (19 aa) of PRLR alone and deletion of
the signal peptide along with mutation of 4P/A of PRLR resulted in
-19 long form and
-19,4P/A PRL-R mutants, respectively
(cytoplasmic forms). Deletion of the signal peptide of PRL-R (mutant
-19) was generated using the cDNA encoding the long form of rat
PRL-R in Bluescript expression vector with PCR amplification of PRL-R
cDNA, in the presence of a 5'-oligonucleotide primer containing an
EcoRI site, the codons for an initiation methionine, and the
first five aa after the signal peptide (aa 2024: QSPPG, see Ref. 43
for the abbreviation) of wild type PRLR
(5'-GTTAACGAATTCATGCAGTCACCACCAGGG-3'); long sense oligonucleotide
contained a sequence complementary to the region encoding amino acids
205210 and an NcoI site (25). Recombinant plasmids were
subsequently subcloned into the EcoRI-XbaI sites
of the expression vector pECE or pcDNA3 (Pharmacia, Piscataway, NJ).
Sequence and orientation of the recombinant plasmids were confirmed by
dideoxynucleotide analysis (44).
Addition of a NLS [of the SV40 large T antigen, PKKKRKV (45)] between
the methionine initiation codon and the first amino acid after the
signal peptide (amino acid 20, see Ref 43) of
-19 mutant long form
using the EcoRI and NcoI restriction sites
resulted in NLS PRLR mutant (nuclear form); addition of a sequence
coding for a Flag epitope in the cDNA of
-19 and NLS mutants has
also been performed for a better visualization of the localization of
these receptors (25).
Truncation of the cytoplasmic tail of the Nb2 form resulted in mutant
Nb2 T322 (8).
Cotransfection of PRLR cDNA and ß-Casein/Luciferase Reporter
Gene in CHO Cells
Biological activities of the different forms and mutants of
PRLRs were analyzed using a functional bioassay based on the
cotransfection of CHO cells with the expression vector pECE containing
cDNAs of the PRLR (and/or mutants PRLR) and a construct that contains a
milk protein (ß-casein) gene promoter fused to the reporter gene of
the firefly luciferase [plasmid ß-casein/luciferase (46)]. CHO
cells were grown on 60-mm culture dishes to 5060% confluency. Twelve
hours before transfection, cells were incubated overnight with GC3
medium (1:1 mixture of DMEM and Hams F12 supplemented with
transferrin (10 µg/ml), insulin (80 mU/ml), glutamine (2.5
mM), and nonessential amino acids. Cells were transfected
by the calcium phosphate procedure (47) with 3 µg long form PRLR/pECE
cDNA plus 3 µg DNA carrier (or 6 µg of the mixture of two PRLR
cDNAs, e.g. the long and short forms of PRLR/pECE cDNA or
two different mutants of PRLR cDNA), 1.5 µg of the fusion gene
construct containing the ß-casein/luciferase coding sequence, and 3
µg pCH110 (ß-galactosidase expression vector, Pharmacia, Bromma,
Sweden). After incubation for 4 h with the calcium phosphate
precipitate, cells were subjected to a 14% (vol/vol) glycerol shock
for 2 min and incubated in the presence of oPRL (50 nM; a
gift of NIDDK, NIH, Bethesda, MD) and dexamethasone (250
nM, Sigma Chemical Co., St. Louis, MO) or dexamethasone
alone for 48 h. Cells were lysed in lysis buffer (Promega,
Madison, WI), and centrifuged at 15,000 rpm for 5 min.
ß-Casein-luciferase activity was measured in the supernatant as
relative light units (Lumat LB 9501, Berthold, Wildbad, Germany); to
correct for differences in transfection efficiencies between plates,
luciferase activity was normalized to ß-galactosidase activity.
Transient Transfection of 293 Fibroblasts and Luciferase
Assay
The human embryonic kidney fibroblast 293 cell line was grown in
DMEM/F12 medium containing 10% FCS. Several hours before transfection,
cells were plated in a rich medium (2/3 DMEM/F12, 1/3 DMEM containing
4.5 g/liter glucose, and 10% FCS). Then, the cells (5 x
105) were cotransfected with 300 ng of plasmid containing
the long and short forms of PRLR cDNA, 500 ng pCH110
(ß-galactosidase), and 100 ng of reporter plasmid that carries the
sequence encoding the luciferase gene, which is under the control of a
six-repeat sequence of the LHRE DNA, followed by the minimal thymidine
kinase promoter (48). Twenty-four hours after transfection, cells were
shifted to serum-free medium with or without 18 nM oPRL.
After 24 h of stimulation, cells were lysed, and luciferase and
ß-galactosidase activation was measured, as described above.
Binding Analysis
COS-7 cells (6070% confluency) were transiently transfected
with the various mutant forms of the PRLR cDNA (5 µg expression
plasmid per 100-mm culture dishes), using the
diethylaminoethyl/dextran-chloroquine procedure.
[125I]hGH was prepared using chloramine T, to a specific
activity of 80140 mCi/mg. The lactogenic hormone human GH binds to
the PRLR with the same affinity as oPRL (19). Forty eight hours after
transfection, cells were scraped and lysed by three freeze-thaw cycles
in 25 mM Tris-HCl, pH 7.410 mM
MgCl2. After centrifugation at 50,000 x g,
100 µg membranes were incubated with 50,000 cpm
[125I]hGH (3 x 108 cpm/µg hGH) in a total volume
of 0.4 ml of 25 mM Tris-HCl, pH 7.410 mM
MgCl2-0.1% BSA. Incubation was carried out at 23 C for
12 h; the microsome suspension was then washed twice with ice-cold
Tris-HCl-0.1% PBS and centrifuged at 4000 x g for 30
min to separate bound [125I]hGH from free ligand.
Radioactivity of the dried pellet was counted in a
-counter.
Specific binding was defined as the difference between total (in the
absence of excess unlabeled ligand) and nonspecific (in the presence of
excess ligand, 1 µg/ml) binding. Scatchard analysis was calculated
from specific binding obtained in the presence of
[125I]hGH with increasing concentrations of unlabeled
oPRL (0.1 ng to 10 µg).
Internalization Studies
Experiments were performed according to the procedure
established by Vincent et al. (47) as follows. Briefly,
COS-7 cells transfected at 7080% confluency using the
diethylaminoethyl/dextran-chloroquine procedure. Forty eight hours
after transfection as described above, cells were washed with HEPES
binding buffer (24 mM HEPES containing 125 mM
NaCl, 4 mM KCl, 1 mM CaCl2, 1.5
mM MgCl2, 2 mM
KH2PO4; pH 7.4) and incubated with
[125I]hGH (100,000 cpm) in the presence or absence of
unlabeled PRL (2.5 µg/well) in 1 ml HEPES binding buffer with 1% BSA
for 6 h at 4 C. The kinetics of internalization were determined
after different periods (060 min) at 37 C. The surface bound
[125I]hGH was removed by exposure to an acid wash
treatment (NaCl, 0.15 M, glycine, 0.05 M; pH
2.5). Cells were lysed with 1 ml NaOH, 1 M, at room
temperature, and lysates radioactivity was counted in a
-counter.
Internalization is expressed as the percentage of intracellular
[125]IhGH with respect to the total specific
cell-associated binding.
Indirect Immunofluorescence
Transfected cells were grown as subconfluent monolayer cultures
in Labtek chambers. For the experiments, cells were harvested for 312
h in culture medium with 0.1% FBS 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 mouse anti-Flag monoclonal antibody (IBI-A
Kodak, New Haven, CT; 10 µg IgG/ml) or mouse monoclonal antibody
against the extracellular domain of the PRLR [U5 (49); 60 µg
IgG/ml] overnight at 4 C, then washed and further incubated for 45 min
with a 1:40 dilution of FITC-conjugated goat anti-IgG, (25). Cells were
then mounted in 50% glycerol in PBS and observed on a Zeiss microscope
(Carl Zeiss, New York, NY). Specificity control experiments included
incubation of cells in the absence of primary and/or secondary
antibodies, or with IgG control monoclonal antibodies. No
immunofluorescence was detected in any of the specificity control
experiments. To confirm the localization of PRLR within the cell,
confocal laser scanning microscopy was performed using a Bio-Rad MRC
600 (Bio-Rad Laboratories, Palo Alto, CA), as previously described
(50).
Immunoprecipitation
293 cells were stimulated with 18 nM oPRL for 10
min. Then, the cells were lysed in 1 ml of lysis buffer (10
nM 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 dimethylsulfonyl fluoride, 1 µg/ml pepstatin A, 2
µg/ml leupeptin, 5 µg/ml aprotinin) for 10 min at 4 C. Next, the
insoluble material was discarded by centrifugation at 12,000 x
g for 5 min. Cell lysates were immunoprecipitated overnight
at 4 C with 2 µl anti-Flag and 15 µl protein A-sepharose before
being washed.
Western Blot Analysis
Proteins were separated on a 7.5% acrylamide gel, transferred
on a polyvinylidene difluoride transfer membrane (Polyscreen, Dupont
NEN, Boston, MA). Blots were incubated with either an
antiphosphotyrosine antibody [4G10, Upstate Biotechnology (Lake
Placid, NY) 1 µg/ml], an anti-PRLR monoclonal antibody (U5 at 5
µg/ml) or an anti-Flag antibody (1 µg/ml) for 2 h at room
temperature and visualized by enhanced chemiluminescence (ECL)
detection (Amersham, Arlington, Heights, IL).
 |
ACKNOWLEDGMENTS
|
---|
We thank G. Geraud (Institut J. Monod, Paris) for help in
confocal laser microscopy and V. Goffin for helpful discussions. Dr.
James Ihle kindly provided the Jak2 cDNA and the National Hormone and
Pituitary Program, NIDDK, provided the oPRL.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Martine Perrot-Applanat, INSERM U 344, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730, Paris Cedex 15, France.
This work was supported by the Institut National de la Santé et
de la Recherche Médicale and The Centre National de la Recherche
Scientifique.
Received for publication August 7, 1996.
Accepted for publication April 8, 1997.
 |
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