Constitutive Activation of the Prolactin Receptor Results in the
Induction of Growth Factor-independent Proliferation and Constitutive
Activation of Signaling Molecules*
Richard C. H.
Lee
§,
Jay A.
Walters
,
Mary E.
Reyland¶, and
Steven M.
Anderson
From the
Department of Pathology, School of Medicine,
and ¶ Department of Basic Science and Oral Biology, School of
Dentistry, University of Colorado Health Sciences Center,
Denver, Colorado 80262
 |
ABSTRACT |
The ability to induce the oncogenic activation of
the human prolactin receptor (PRLR) was examined by deleting 178 amino
acids of the extracellular ligand-binding domain. Expression of this deletion mutant in the interleukin-3 (IL-3)-dependent
murine myeloid cell line 32Dcl3 resulted in the induction of growth
factor-independent proliferation. Parental 32Dcl3 cells proliferated
only in the presence of exogenous murine IL-3 (mIL-3), while 32Dcl3
cells transfected with the long form of the human PRLR were able to proliferate in response to mIL-3, ovine prolactin, or human PRL. Cells
expressing the
178 deletion mutant contained numerous
phosphotyrosine-containing proteins in the absence of stimulation with
either mIL-3 or ovine prolactin. Growth factor stimulation increased
the number of proteins phosphorylated and the intensity of
phosphorylation. These proteins included constitutively phosphorylated
Janus kinase 2, signal transducer and activator of transcription 5, and
SHC. Activated extracellular signal-regulated kinases 1 and 2 (ERK1 and
ERK2) were observed in unstimulated 32Dcl3 cells expressing the
178 mutant. Likewise, transfection of Nb2 cells with the
178 deletion mutant induced growth factor-independent proliferation and constitutive activation of Janus kinase 2, ERK1, and ERK2. In addition to the induction of a growth factor-independent state, the expression of the
178 deletion mutant also suppressed the apoptosis that occurs when
32Dcl3 cells are cultured in the absence of growth factors such as
IL-3. These data suggest that the constitutive activation of the PRLR
can be achieved by deletion of the ligand binding domain and that this
mutation leads to the oncogenic activation of the receptor as
determined by the ability of the receptor to induce growth
factor-independent proliferation of factor-dependent hematopoietic cells.
 |
INTRODUCTION |
Ligand-induced oligomerization of growth factor receptors is
critical in regulating the proliferation and differentiation of cells.
Dimerization of transmembrane receptor tyrosine kinases leads to the
activation of their intrinsic kinases, phosphorylation of the receptor
molecules themselves, and the subsequent activation of secondary
signaling molecules. Members of the cytokine receptor superfamily lack
intrinsic kinase activity; however, several different tyrosine kinases
become activated following ligand binding. These kinases phosphorylate
the receptors as well as numerous other substrates, leading to the
activation of many of the same signaling pathways that lie downstream
of receptor tyrosine kinases. Cytokine receptors generally activate two
classes of tyrosine kinases: one or more members of the Janus family of
tyrosine kinases as well as one or more members of the src
family of tyrosine kinases. Janus kinases are directly responsible for
the phosphorylation and activation of a class of transcription factors
referred to as STATs1 (1). It
is not clear, however, which family of tyrosine kinases are required
for the activation of the Ras/Raf/MAP kinase cascade or
phosphatidylinositol 3-kinase.
Given the critical role of these receptors in regulating cellular
proliferation, it is not surprising that mutations that result in their
constitutive activation are also oncogenic. At least four
constitutively activated transmembrane receptor tyrosine kinases have
been transduced by oncogenic retroviruses: v-Erb B, v-Fms, v-Kit, and
v-Ros. Erb-B, c-Fms, and c-Kit represent the receptors for epidermal
growth factor, colony-stimulating factor-1, and stem cell factor,
respectively (2-6). These receptors can be activated by point
mutations in the ligand binding domain, the transmembrane domain,
and/or the intracellular domain and by deletion of the majority of the
extracellular domain (2, 7-12).
To date, only a single cytokine receptor has been discovered to be
transduced by an oncogenic retrovirus; the v-mpl oncogene in
the murine myeloproliferative leukemia virus is a truncated version of
the receptor for thrombopoietin (13-15). Activating mutations have
also been described in two other members of the cytokine receptor
superfamily: the erythropoietin receptor and the
c subunit (
common subunit) of the receptors for IL-3, IL-5, and
granulocyte-macrophage colony-stimulating factor (16-21).
The current study was undertaken to determine whether deletion of the
extracellular domain of the prolactin receptor (PRLR) would lead to its
constitutive activation and the ability to induce growth
factor-independent proliferation of factor-dependent
hematopoietic cell lines. In this report, we demonstrate that the
deletion of a 178-amino acid region of the extracellular domain of the
PRLR resulted in its constitutive activation and the induction of
growth factor-independent proliferation. The constitutive
phosphorylation of multiple proteins including JAK2, STAT5, and SHC was
observed in addition to growth factor-independent activation of MAP
kinase. Although previous studies by Gourdou et al. (22)
have demonstrated that deletion of a region of the extracellular domain
of the rabbit PRLR resulted in the constitutive activation of the
receptor as determined by the PRL-independent activation of
-casein
transcription, these investigators did not demonstrate the activation
of any signaling molecules, the induction of growth factor-independent proliferation, or the suppression of apoptosis. Our results are discussed in the context of signal transduction by the PRLR and its
role in oncogenesis.
 |
MATERIALS AND METHODS |
Cell Culture--
The 32D clone 3 (32Dcl3) cell line was
obtained from Dr. Joel Greenberger (University of Pittsburgh,
Pittsburgh, PA) (23), and it was maintained as described by Anderson
and Jorgensen (24). Recombinant murine IL-3 was obtained from
Collaborative Biomedical Products, Inc. (Bedford, MA). Human and ovine
PRL (Lots AFP-3855A and AFP-10677C, respectively) were obtained from
the National Hormone and Pituitary Program (Rockville, MD). Recombinant
human PRL (rhPRL) was purchased from Genzyme (Cambridge, MA) or R & D
Systems (Minneapolis, MN).
The 32D/hPRLR and 32D
178 cell lines were generated by introducing
the plasmid DNAs of interest into the 32Dcl3 cell line with a
Cell-Porator (1,000 V/cm at 800 microfarads) (Life Technologies, Inc.).
Electroporation chambers used had a 0.4-cm gap between the electrodes,
and cells were resuspended in Dulbecco's modified Eagle's medium that
was at room temperature without any additional supplements . Both the
hPRLR and the
178 deletion mutant cDNAs were subcloned into the
pcDNA3 vector (Invitrogen, Carlsbad, CA), which contains the
neomycin resistance marker as a dominant selectable marker. Following
electroporation, the cells were cultured in the presence of IL-3 for
2-3 days, and the transfected cells were selected by the addition of
1.0 mg/ml G418 (Life Technologies). G418-resistant cells were allowed
to expand with IL-3 for 3 days, after which the drug was removed. Cells
were then cultured in oPRL in the case of the 32D/hPRLR or in the
absence of any growth factors in the case of 32D
178 cells. Single
cell clones were then isolated following growth of the cells at
limiting dilution in semisolid media containing 0.6% SeaPlaque agarose
(FMC Corp., Freeport, ME). Isolated colonies were picked from soft
agar, expanded in liquid culture, and used in the described experiments.
The Nb2 cell line was obtained from Lee-Yuan Yu-Lee, and the cells were
maintained as described previously (25). Transfected clones of Nb2
cells were produced in an identical manner to that described above.
Expression Vector Construction--
A cDNA clone of the
human PRLR was obtained from Paul Kelly (INSERM, Paris, France). The
full-length cDNA was removed by digesting the plasmid with
EcoRI, gel-purifying the 2,556-base pair insert, and
inserting the DNA into the EcoRI site of pcDNA3
(Invitrogen). The
178 deletion mutant was created by digesting the
full-length cDNA with both BglII and NcoI,
filling in the overhangs with Klenow DNA polymerase (Life
Technologies), and religating the plasmid DNA. This results in the
deletion of 534 base pairs, corresponding to a deletion of 178 amino
acids. Deletion of the desired fragment was confirmed by DNA sequence
analysis. The complete cDNA fragment containing this deletion was
removed by digestion with EcoRI and cloned into the
EcoRI site of pcDNA3. Correct orientation of the cDNA insert in pcDNA3 was confirmed by diagnostic restriction enzyme analysis and by DNA sequence analysis from both ends of the
multiple cloning site of pcDNA3.
The FLAG epitope tag was added to the C-terminal end of the hPRLR and
the
178 deletion mutant cDNAs using polymerase chain reaction.
The upstream primer used in this reaction was
5'-TGTGCTTGGAATTCCCCG-3', and the downstream primer was
5'-CCGAATTCGGGTCACTTGTCGTCGTCATCCTTGTAGTCGTGAAAGGAGTG-3'.
RNA Isolation and Northern Blot Analysis--
RNA was isolated
from each cell line using TRIzol Reagent (Life Technologies) used
according to the manufacturer's recommendation. Twenty micrograms of
total cellular RNA were size-separated in a 1.2% agarose gel
containing 2.2 M formaldehyde. The resolved RNAs were
electrotransferred to Nytran (Schleicher and Schuell), and the RNAs
were cross-linked to the membrane with a Stratagene Stratalinker (La
Jolla, CA). 32P-Labeled probes to the hPRLR and to GAPDH
were prepared by random priming in the presence of
[
-32P]dCTP (Amersham Pharmacia Biotech; 800 Ci/mmol).
The rat GAPDH cDNA clone was originally obtained from Dr. Kenneth
Marcu (State University of New York at Stony Brook).
Immunoprecipitation and Immunoblotting--
Immunoprecipitation
and immunoblotting were performed as described previously (24, 26).
Cells to be immunoprecipitated were cultured overnight in McCoy's 5A
media supplemented with 5% charcoal-stripped serum (HyClone; Orham,
UT) to reduce the background level of tyrosine-phosphorylated proteins.
Cells were then resuspended in media containing 100 units/ml
recombinant murine IL-3, 100 nM (2.23 µg/ml) ovine PRL,
or 0.5-1.0 µg/ml rhPRL for the indicated periods of time. Cells were
lysed in either radioimmune precipitation buffer (150 mM
NaCl, 50 mM Tris (pH 7.4), 2 mM EGTA, 1%
Triton X-100, 1 mM sodium orthovanadate) or extraction
buffer (50 mM NaCl, 10 mM Tris (pH 7.4), 5 mM EDTA, 50 mM NaF, 1% Triton X-100, 1 mM sodium orthovanadate). Both lysis buffers were
supplemented with 100 units/ml aprotinin (Calbiochem). Agarose-conjugated monoclonal anti-phosphotyrosine antibody 4G10, rabbit anti-JAK2, and rabbit anti-ERK1/2 were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Unconjugated monoclonal anti-phosphotyrosine antibody 4G10 was the generous gift of Brian Druker (Oregon Health Sciences Center, Portland, OR). Rabbit anti-SHC (catalogue no. S14630) and a monoclonal antibody directed against SHC
(catalogue no. S14620) were obtained from Transduction Laboratories (Lexington, KY). Anti-ACTIVE MAP kinase, a polyclonal antibody directed
against the activated version of ERK2 that shows some cross-reaction
with activated ERK1, was obtained from Promega (Madison, WI). Rabbit
anti-STAT5 antiserum was the gift of Andrew Larner (National Institutes
of Health, Bethesda, MD), and a monoclonal antibody directed against
STAT5 was obtained from Transduction Laboratories. Agarose-conjugated
anti-FLAG M2 monoclonal antibody was obtained from Scientific Imaging
Systems-Kodak (New Haven, CT), anti-FLAG M2 monoclonal antibody was
from Sigma, and rabbit anti-FLAG polyclonal IgG was from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Preparation of the monoclonal
antibody directed against the
-subunit of the murine IL-3 receptor
has been described (27). Immunoprecipitated proteins were resolved on 8 or 10% SDS-polyacrylamide gels and electrotransferred to Immobilon
membranes (Millipore Corp., Bedford, MA). Immunoblotting was conducted
as described by Anderson et al. (26) with the ECL system according to
the manufacturer's recommendations (Amersham Pharmacia Biotech).
Proliferation Assays--
Cells (32Dcl3, 32D
178, and
32D/hPRLR) were resuspended in McCoy's 5A medium supplemented with or
without growth factors as indicated. The number of viable cells was
determined at the different time points by counting the number of cells
that excluded trypan blue.
Preparation of Membrane Fractions--
Cells (20 × 106) were washed two times with 5 ml of ice-cold
phosphate-buffered saline. Cells were resuspended in 1 ml of cold membrane lysis buffer (20 mM Tris-HCl (pH 8.0), 2 mM MgCl2, 1 mM EDTA, 1 mM
-mercaptoethanol) supplemented with 1 mM
sodium orthovanadate and 100 units/ml aprotinin. Cells were broken by passage through a 31-gauge needle and then centrifuged for 5 min at
1,000 rpm at 4 °C to remove nuclei. The supernatant fraction was
then centrifuged for 30 min at 15,000 rpm, 4 °C to obtain a membrane
fraction. The supernatant was carefully removed, and the pellet was
resuspended in 100 µl of membrane lysis buffer and passed once again
through a 31-gauge needle. Membrane proteins (100 µg) were resolved
on 8% polyacrylamide gel, transferred to Immobilon membranes, and
immunoblotted using anti-FLAG M2 monoclonal antibody (Sigma) or rabbit
anti-FLAG probe polyclonal IgG (Santa Cruz Biotechnology).
Analysis of DNA Fragmentation Due to Apoptosis--
Cells were
pelleted by centrifugation and washed once with phosphate-buffered
saline. Cells were lysed in 400 µl of DNA lysis buffer (50 mM Tris, pH 7.5, 10 mM EDTA, 0.5% SDS, 100 µg/ml RNase A, 4 µg/ml proteinase K) and incubated at 37 °C for
6 h. DNAs were extracted from cell lysates with 400 µl of
phenol/chloroform/isoamyl alcohol (25:24:1) mix. The salt concentration
was raised to 0.25 M sodium acetate, and the DNA was
precipitated by the addition of 1 ml of 100% EtOH. Following
incubation at
20 °C, the DNAs were pelleted and then resuspended
with 50 µl of TE (10 mM Tris, pH 7.4, 1 mM
EDTA) with 20 µg/ml RNase A. DNAs were quantitated spectroscopically,
and DNA fragmentation was examined by running 10 µg of DNA on a 1.5%
agarose gel.
 |
RESULTS |
Construction of the
178 Deletion Mutant--
Mutation or
deletion of the ligand binding domain of numerous cytokine receptor
family members has been shown to result in their constitutive
activation (7). To determine whether this would also hold true for the
PRLR, a region of the extracellular domain of the hPRLR was deleted,
and the ability of this deletion mutant to induce growth
factor-independence was assessed. A cDNA clone of the hPRLR was
digested with BglII and NcoI, resulting in a loss
of 178 amino acids in the extracellular domain of the receptor. These
restriction sites were chosen because they were unique restriction
sites, they allowed removal of the majority of the extracellular
ligand-binding domain, they retained the WSXWS motif, and
they maintained the proper reading frame when the overhangs were filled
and religated. This deleted version of the hPRLR will be referred to as
hPRLR
178 or
178. The region of the hPRLR deleted is indicated in
Fig. 1. The cDNAs of both the wild
type hPRLR and the hPRLR
178 mutant were inserted into the eukaryotic
expression vector pcDNA3. Both DNA molecules were introduced into
the growth factor-dependent murine myeloid cell line
32Dcl3, and G418-resistant clones were isolated and expanded for use in
the described studies. The 32Dcl3 cell line is absolutely dependent
upon the presence of an exogenous source of mIL-3 for proliferation and
viability (28). When cultured in the absence of IL-3 for 12-16 h, one
can detect DNA fragmentation typical of cells undergoing apoptosis, and
by 48-60 h no viable cells remain. Cells transfected with the hPRLR
construct (32D/hPRLR cells) were capable of proliferating in mIL-3,
hPRL, or oPRL; however, the parental 32Dcl3 cells only proliferated in
mIL-3. In contrast, cells transfected with the hPRLR
178 mutant
(32D
178) were capable of proliferating in the absence of an
exogenous growth factor. Single cell-derived clones of both 32D/hPRLR
and 32D
178 cells were obtained by plating the cells in semisolid
media containing the appropriate growth factors and picking individual
colonies for expansion in liquid media. A second set of constructs was made in which the FLAG epitope tag was added to the C-terminal end of
the hPRLR and the
178 deletion mutant. All studies reported in this
paper used these single cell-derived clones expressing either the
untagged or FLAG-tagged molecules. Identical results were obtained with
both the untagged and FLAG-tagged molecules; however, the majority of
the data shown below was generated using cells expressing the untagged
molecules.

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 1.
Structure of the human prolactin receptor and
the 178 deletion mutant. The region
between amino acid 9 (BglII site) and amino acid 187 (NcoI site) was deleted from the full-length human PRLR to
generate the 178 deletion mutant. The location of the conserved
WSXWS motif is indicated by the asterisk. The
filled box indicates the location of the
transmembrane domain (amino acids 211-234). The location of the three
inserted KpnI sites used by Gourdou et al. (22)
to generate deletion mutants of the rabbit PRLR are indicated at the
bottom for purposes of comparison.
|
|
The hPRLR
178 Deletion Mutant Induces Growth Factor-independent
Proliferation--
The proliferation of 32Dcl3, 32D/hPRLR, and
32D
178 cells were examined in media supplemented with 7.5% fetal
calf serum, media supplemented with 7.5% fetal calf serum and 10%
WEHI-3 conditioned media (a source of IL-3), or media supplemented with
7.5% fetal calf serum and 200 ng/ml oPRL (Fig.
2). All three cell lines proliferated in
media supplemented with WEHI-3 conditioned media; by 72 h, the
cell number of each cell line rose from 4 × 106 cells
to approximately 25-35 × 106 cells. There was no
significant difference in the proliferation rate of cells transfected
with either hPRLR or the
178 mutant of the hPRLR (Fig.
2A). When cultured in media containing 7.5% fetal calf
serum but lacking mIL-3, only the 32D
178 cells increased in number;
the number of viable 32Dcl3 cells decreased, while the number of viable
32D/hPRLR cells remained approximately the same (Fig. 2B).
We suspect that the serum used in this study must have contained low
levels of PRL, since the 32D/hPRLR cells remained viable, while the
32Dcl3 cells died during the first 3 days in culture. The 32D/hPRLR
cells die after 5-6 days in culture in the absence of exogenously
provided PRL (data not shown). We suspect that low levels of PRL
present in fetal calf serum might support the viability of 32D/hPRLR
cells for longer than that of the 32Dcl3 cells, which require IL-3 for
proliferation and viability. Examination of the growth of the same
cells in media supplemented with 7.5% fetal calf serum plus 200 ng/ml
oPRL revealed that the parental 32Dcl3 cells decreased in number, while
both the 32D
178 and the 32D/hPRLR cells increased in number (Fig.
2C). The results shown are an average of three different
studies. Similar data were obtained with five different independent
clones of each cell type.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 2.
Growth of 32Dcl3,
32D 178, and 32D/hPRLR cells under different
conditions. 32Dcl3 ( ), 32D 178 ( ), and 32D/hPRLR ( )
cells were grown under different medium conditions. A, the
cells were grown in media supplemented with 10% WEHI-3 cell
conditioned media; B, the cells were cultured in media
without any additional growth factors; C, the cells were
grown in media supplemented with 200 ng/ml oPRL. Cell counts were
determined at the indicated time intervals of 0, 8, 16, 24, 36, 48, and
72 h. The graphs represent the mean of three different assays.
Experimental error was <10%.
|
|
hPRLR and hPRLR
178 Proteins Are Expressed in Transfected 32Dcl3
Cells--
Expression of the hPRLR or the
178 deletion mutant in
the transfected 32Dcl3 cells was demonstrated by Northern blot analysis (Fig. 3, A and B).
Twenty micrograms of total cellular RNA was resolved by agarose gel
electrophoresis, the separated RNAs were transferred to a nylon
membrane, and the blot was probed with either a probe for the human
PRLR cDNA or a GAPDH cDNA probe to demonstrate equal loading of
the gel. Although there was no RNA species detected in the parental
32Dcl3 cells, a single species of RNA was detected in the cells
transfected with either the hPRLR or the
178 expression vector (Fig.
3A). Five clones of cells that were transfected with the
FLAG-tagged hPRLR molecule had equivalent amounts RNA detected with the
hPRLR probe (Fig. 3A, lanes 1-5). The
five clones of 32Dcl3 cells transfected with the FLAG-tagged
178
deletion mutant had similar amounts of RNA detected with the PRLR
probe; however, it appeared that there was less RNA than in the cells
expressing the hPRLR molecule (Fig. 3A, lanes
7-11). The differences in the sizes of the RNAs observed in
the 32D/hPRLR versus the 32D
178 cells were consistent
with the size of the deletion that had been introduced into the hPRLR cDNA molecule. Reprobing the blot in Fig. 3A with the GAPDH probe revealed that roughly equal amounts of RNA were loaded in all but the
32Dcl3 cell sample (Fig. 3B). There clearly was more GAPDH RNA present in lane 6, indicating that more
32Dcl3 cell RNA was loaded in the gel shown in Fig. 3.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 3.
Expression of the hPRLR and
178 mRNA and proteins in transfected 32Dcl3
cells. A, total cellular RNA was isolated from 32Dcl3
(lane 6), five clones of 32Dcl3 cells transfected
with a FLAG-tagged version of the hPRLR (lanes
1-5), and five clones of 32Dcl3 cells transfected with a
FLAG-tagged version of the 178 deletion mutant (lanes
7-11). The RNAs were size-separated by agarose gel
electrophoresis and transferred to a nylon membrane, and the membrane
was probed with a cDNA probe for the hPRLR. The positions of the 18 and 28 S ribosomal RNAs are indicated on the left.
B, the same Northern blot was reprobed with a cDNA probe
for GAPDH to demonstrate that equal levels of cellular RNA were loaded.
C, membrane fractions of 32Dcl3 and clones of 32Dcl3 cells
transfected with an expression vector encoding a FLAG-tagged hPRLR were
prepared. One hundred micrograms of membrane proteins were resolved on
an 8% polyacrylamide gel, and the immunoblot was probed with an
anti-FLAG antibody. D, whole cell lysates were prepared from
32Dcl3 and clones of 32Dcl3 cells transfected with an expression vector
encoding a FLAG-tagged 178 deletion mutant. One milligram of the
indicated cell lysates were precleared by the addition of 1 µg of a
mouse monoclonal antibody directed against the -subunit of the IL-3
receptor (27), which was bound to Omnisorb (Calbiochem). After removing
any bound proteins, agarose-conjugated anti-FLAG M2 antibody was added
to the precleared lysate. The bound proteins were resolved by
electrophoresis, and the immunoblot was probed with an anti-FLAG
antibody. The cell lines examined are identified at the top
of each lane, and lane numbers are indicated at the
bottom of each lane. The positions of prestained
protein markers are indicated on the left side of
C and D.
|
|
To demonstrate the presence of the hPRLR and
178 proteins in the
transfected cells analyzed by Northern blot analysis, we used
immunoblot analysis with an anti-FLAG epitope tag monoclonal antibody.
In Fig. 3C, the expression of the hPRLR protein in the transfected cells is demonstrated. A membrane fraction was isolated from these 32Dcl3 and 32D/hPRLR cells, 100 µg of membrane protein was
resolved by SDS gel electrophoresis, and the blot was subjected to
immunoblotting with anti-FLAG antibody. Clones 6, 9, and 10 of the
32D/hPRLR cells appeared to have nearly equivalent levels of the hPRLR
protein, while clones 7 and 8 appeared to have lower levels of the
hPRLR protein (Fig. 3C, lanes
13-17).
We were also able to detect expression of the
178 protein in
transfected cells by immunoblotting (Fig. 3D). Whole cell
lysates of 32D
178 cells were prepared and were precleared with an
irrelevant antibody (a monoclonal antibody directed against the
subunit of the IL-3 receptor) (27). The lysates were then
immunoprecipitated with agarose-conjugated anti-FLAG M2 antibody, and
the immunoprecipitated proteins were resolved on an SDS gel.
Immunoblotting was with an unconjugated anti-FLAG monoclonal antibody.
Approximately equal amounts of the
178 protein were present in
all four clones analyzed, although clone 12 might have slightly less
protein (Fig. 3D, lanes 19-22). The
sizes of the proteins detected in the different cell lines were
consistent with that expected. Since different approaches were used to
detect the presence of the FLAG-tagged protein molecules in the two
different cell types, we cannot directly compare the amount of
178
protein with that of the hPRLR. For reasons that we do not understand
at this time, we were not able to detect the hPRLR by
immunoprecipitating whole cell lysates, nor were we able to detect the
178 protein in membrane fractions prepared from 32D
178 cells
(data not shown).
Constitutive Receptor Activation Results in Altered Patterns of
Tyrosine Phosphorylation--
Stimulation of the PRL-responsive Nb2
cell line has been shown to result in the phosphorylation of multiple
proteins on tyrosine residues, including the receptor itself (29). The
phosphorylation of cellular proteins was examined in cells expressing
the hPRLR
178 mutant by anti-phosphotyrosine immunoblot analysis. The
pattern of tyrosine-phosphorylated proteins was examined following
stimulation of 32Dcl3, 32D/hPRLR, and 32D
178 cells with either mIL-3
or oPRL for 0, 5, or 15 min. Stimulation of either 32Dcl3 or 32D/hPRLR cells with mIL-3 resulted in the rapid appearance of proteins with
molecular masses corresponding to 120-150, 95, 52, 46, and 42 kDa
(Fig. 4, lanes 1-3
and 7-9). The 120-150-kDa region includes many different
proteins including the
-subunit of the IL-3 receptor (30-32), JAK2
(33), CBL (26), SHIP (34, 35), and
CAS.2 The 52- and 46-kDa
proteins probably represent two of the isoforms of SHC (36), and the
42-kDa protein probably represents ERK2 (37) (see below). The observed
95-kDa protein does not represent STAT5 but instead represents an
unidentified molecule whose phosphorylation is detected in a variety of
cells stimulated with many different growth factors (data not shown).
We do not routinely observe phosphorylated STAT molecules using the
approach described in Fig. 4 (data not shown). In contrast to both
32Dcl3 and 32D/hPRLR cells, numerous tyrosine-phosphorylated proteins
were observed in unstimulated 32D
178 cells (Fig. 4, lane
4). Stimulation of 32D
178 cells with mIL-3 appeared to
increase the extent of phosphorylation of both SHC and ERK2 (Fig. 4,
lanes 4-6).

View larger version (86K):
[in this window]
[in a new window]
|
Fig. 4.
Tyrosine-phosphorylated
proteins are observed in unstimulated
32D 178 cells. The pattern of
phosphotyrosine-containing proteins was examined in 32Dcl3 cells
(lanes 1-3 and 10-12), 32D 178
clone 2 cells (lanes 4-6 and 13-15),
and 32D/hPRLR clone 1 cells (lanes 7-9 and
16-18) stimulated with either 100 units/ml mIL-3
(lanes 1-9) or 100 nM oPRL
(lanes 10-18). Cells were stimulated for 0, 5, or 15 min prior to lysis, and the time of stimulation is indicated at
the top of each lane. Cells were
immunoprecipitated with the anti-phosphotyrosine monoclonal antibody
4G10, and the precipitated proteins were resolved on an 8%
polyacrylamide gel. Immunoblotting was with anti-phosphotyrosine
monoclonal antibody 4G10. The positions of prestained molecular mass
markers are indicated on the left. Lane numbers are
indicated at the bottom of each lane.
|
|
As would be expected, since these cells lack the PRLR, stimulation of
32Dcl3 cells with 100 nM oPRL did not result in the appearance of tyrosine-phosphorylated proteins (Fig. 4,
lanes 10-12). Lanes 10-18
of Fig. 4 were exposed longer than lanes 1-9, to
allow for the detection of PRL-induced phosphorylation of proteins in
lanes 16-18; however, this resulted in a higher
background. This is most evident in lanes 10-15
of Fig. 4. Despite this higher background, it is clear that PRL did not
stimulate an increase in the phosphotyrosine-containing proteins in
32Dcl3 cells (Fig. 4, lanes 10-12). Stimulation
of the 32D/hPRLR cells with oPRL resulted in a significant increase in
the level of phosphorylated proteins (Fig. 4, lanes
16-18); most notable were proteins with molecular masses
expected to correspond to SHC and JAK2. We do not know why there are
more tyrosine-phosphorylated proteins present in unstimulated 32D/hPRLR
cells than in unstimulated 32Dcl3 cells. Consistent with the results
described above, we observed the constitutive phosphorylation of
proteins with sizes that correlate with known signaling molecules in
unstimulated 32D
178 cells. We were surprised to observe an increase
in the phosphorylation of proteins in the 120-150-kDa range following
stimulation of these cells with oPRL (Fig. 4, lanes
13-15). Based upon the size of this protein, we would
expect that this protein would correspond to JAK2 (see below). We
believe that the increase in the phosphorylation of this band is due to
the presence of another growth factor in the preparations of oPRL used
in these studies, since rhPRL did not stimulate an increase in JAK2
phosphorylation (see below); however, in studies not shown here, oPRL
did increase the phosphorylation of JAK2 (data not shown). These data
demonstrate that the expression of the
178 mutant of the hPRLR in
32Dcl3 cells results in the constitutive phosphorylation of numerous
proteins, and consistent with data in Fig. 2, stimulation with mIL-3
resulted in the increased phosphorylation of several proteins.
Consistent results were obtained in four different studies, and similar
results were also obtained with the original pool of uncloned 32D
178
and 32D/hPRLR cells studied prior to isolation of single cell-derived clones.
Is the JAK/STAT Pathway Constitutively Activated in 32D
178
Cells?--
The binding of either IL-3 or PRL to its respective
receptor has been shown to result in the rapid activation of JAK2
tyrosine kinase (33, 38-40). Activation of Janus family tyrosine
kinases, such as JAK2, results in the phosphorylation and activation of a family of proteins known as STATs. STAT molecules are cytosolic proteins that become tyrosine-phosphorylated following activation of
Janus family kinases, dimerize, and translocate to the nucleus, whereupon they bind to specific DNA elements and activate transcription (1). To determine whether JAK2 was constitutively activated in
32D
178 cells, anti-JAK2 immunoprecipitates were subjected to
anti-phosphotyrosine immunoblotting (Fig.
5). The tyrosine phosphorylation of JAK2
has been associated with the catalytic activation of the enzyme.
Stimulation of 32Dcl3 cells with mIL-3 resulted in the phosphorylation
of JAK2 (Fig. 5, lanes 1-3). In a similar
fashion, stimulation of 32D/hPRLR cells with mIL-3 resulted in the
phosphorylation of JAK2, although the signal was not as robust as that
seen with 32Dcl3 cells (Fig. 5, lanes 7-9). In the absence of mIL-3 stimulation, tyrosine-phosphorylated JAK2 was
readily detectable in 32D
178 cells, and mIL-3 stimulation increased
the phosphorylation of JAK2 approximately 4-5-fold, as indicated by
densitometric measurements (Fig. 5, lanes
4-6).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 5.
JAK2 is tyrosine-phosphorylated in the
absence of growth factor stimulation in 32D 178
cells. A, JAK2 was immunoprecipitated from 32Dcl3 cells
(lanes 1-3 and 10-12), 32D 178
clone 2 cells (lanes 4-6 and 13-15),
and 32D/hPRLR clone 1 cells (lanes 7-9 and
16-18) stimulated with either 100 units/ml mIL-3
(lanes 1-9) or 1 µg/ml rhPRL (lanes
10-18). Cells were stimulated for 0, 5, or 15 min prior to
lysis, and the time of stimulation is indicated at the top
of each lane. Cell lysates were immunoprecipitated with a
polyclonal antibody specific for JAK2, and the precipitated proteins
were resolved on an 8% polyacrylamide gel. Immunoblotting was with
anti-phosphotyrosine monoclonal antibody 4G10. The position of JAK2 is
indicated by the arrow on the right.
B, equal levels of JAK2 protein were demonstrated by running
100 µg of whole cell lysate on an 8% polyacrylamide gel and
immunoblotting the proteins with anti-JAK2 antibody. Lane numbers are
indicated at the bottom of each lane.
|
|
Stimulation of 32D/hPRLR cells with 1.0 µg/ml rhPRL resulted in the
rapid phosphorylation of JAK2, which was not observed in 32Dcl3 cells
stimulated with rhPRL (Fig. 5, lanes 10-12
versus lanes 16-18). This concentration of rhPRL
was chosen based upon the dose reported by the manufacturer to provide
maximal stimulation of T cell proliferation. Tyrosine-phosphorylated
JAK2 was observed in unstimulated 32D
178 cells, and stimulation with
rhPRL caused a decrease in the phosphorylation of JAK2 after 5-15 min
of stimulation (Fig. 5, lanes 13-15). The level
of JAK2 was similar in all cell lines examined and was not affected by
stimulation with either mIL-3 or rhPRL (Fig. 5B).
The constitutive activation of JAK2 in 32D
178 cells suggested to us
that the constitutive activation of STAT molecules might also be
observed in these cells. STAT5 was immunoprecipitated with a polyclonal
antibody specific for STAT5, and its phosphorylation was examined by
anti-phosphotyrosine immunoblotting. Consistent with the results shown
in Fig. 5, tyrosine-phosphorylated STAT5 was present in unstimulated
32D
178 cells (Fig. 6A, lane
4). Little or no phosphorylated STAT5 was detected in unstimulated
32Dcl3 or 32D/hPRLR cells (Fig. 6A, lanes
1 and 7). Although stimulation of 32Dcl3 and
32D/hPRLR cells with IL-3 resulted in an increase in the amount of
phosphorylated STAT5, we did not observe a corresponding increase in
STAT5 phosphorylation in the 32D
178 cells (Fig. 6A, lanes 4-6). Stimulation of 32D/hPRLR cells with
rhPRL resulted in a robust increase in STAT5 phosphorylation,
consistent with that observed following stimulation of the same cells
with mIL-3 (Fig. 6A, lanes 16-18). No
tyrosine-phosphorylated STAT5 was detected in unstimulated or
rhPRL-stimulated 32Dcl3 cells, consistent with the absence of the PRLR
in these cells. Tyrosine-phosphorylated STAT5 was detected in
unstimulated 32D
178 cells, and stimulation with rhPRL resulted in a
decrease in the amount of phosphorylated STAT5 (Fig. 6A,
compare lanes 13-15). Although this result is consistent with decreased phosphorylation of JAK2 in these cells described in Fig. 5, the mechanism behind the decreased phosphorylation of both JAK2 and STAT5 in 32D
178 cells stimulated with rhPRL is not
clear. There is no reason to suspect that rhPRL would be down-regulating the levels of the
178 protein; however, that remains
a possibility. It is clear that rhPRL does not induce a decrease in the
amount of either JAK2 or STAT5 protein. The levels of STAT5 protein
present in unstimulated and IL-3-stimulated 32Dcl3, 32D
178, and
32D/hPRLR cells were not significantly different (Fig. 6,
bottom panel).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 6.
STAT5 is tyrosine-phosphorylated in the
absence of growth factor stimulation in 32D 178
cells. A, STAT5 was immunoprecipitated from 32Dcl3
cells (lanes 1-3 and 10-12),
32D 178 clone 2 cells (lanes 4-6 and
13-15), and 32D/hPRLR clone 1 cells (lanes
7-9 and 16-18) stimulated with either 100 units/ml mIL-3 (lanes 1-9) or 1.0 µg/ml rhPRL
(lanes 10-18). Cells were stimulated for 0, 5, or 15 min prior to lysis, and the time of stimulation is indicated at
the top of each lane. Cell lysates were
immunoprecipitated with a polyclonal antibody specific for STAT5, and
the precipitated proteins were resolved on an 8% polyacrylamide gel.
Immunoblotting was with anti-phosphotyrosine monoclonal antibody 4G10.
The position of STAT5 is indicated by the arrow on the
right. B, the blot shown in A was
reprobed with anti-STAT5 antibody to demonstrate the amount of STAT5
protein in each sample. Lane numbers are indicated at the
bottom of each lane.
|
|
Activation of Mitogen-activated Protein Kinase in 32D
178
Cells--
Cytokine stimulation has been reported to activate both
ERK1 and ERK2, members of the MAP kinase family; this is true for both
PRL (41, 42) and IL-3 (37). Catalytic activation of both ERK1 and ERK2
requires the phosphorylation of two amino acids, Thr183 and
Tyr185 (43), and the activation of ERKs can be examined
through the use of an antibody that recognizes these dually
phosphorylated forms of these enzymes. The phosphorylation of MAP
kinase was examined in stimulated and unstimulated 32Dcl3, 32D/hPRLR,
and 32D
178 cells by immunoblotting with the anti-ACTIVE MAP kinase antibody, and the levels of ERK1 and ERK2 were examined by
immunoblotting with a polyclonal antibody directed against both ERK1
and ERK2 (Fig. 7). Stimulation of 32Dcl3
and 32D/hPRLR cells with mIL-3 resulted in the activation of both ERK1
and ERK2 as indicated by its reactivity with anti-ACTIVE MAP kinase
antibody (Fig. 7, lanes 1-3 and
7-9). In the absence of IL-3 stimulation, no activated ERKs
were detected in either 32Dcl3 or 32D/hPRLR cells. In contrast, 32D
178 cells possessed activated ERKs in the absence of cytokine stimulation (Fig. 7, lanes 4-6 and
13-15). Stimulation of 32D
178 cells with either mIL-3
resulted in an increase in the amount of activated ERK1 or ERK2;
however, stimulation of 32D
178 clone 2 cells with rhPRL did not
result in a comparable increase in ERK activation (Fig. 7). Although
there might be a slight increase in the amount of activated ERKs
present after 15-min stimulation with rhPRL, it represents a relatively
small increase, and it is not clear whether it is significant. When the
amounts of activated ERK1 and ERK2 were examined after 30 min of
stimulation with mIL-3, oPRL, or rhPRL, the amount of phosphorylated
ERK1 and ERK2 decreased in 32Dcl3 and 32D/hPRLR cells, and there was no
change in the amount of activated ERK in the 32D
178 cells (data not
shown). There was no difference in the amounts of ERK1 and ERK2 among the different cell lines (Fig. 7, bottom panel).
This indicates that in addition to JAK2, ERKs are constitutively
activated in 32D
178 cells. Furthermore, like the activation of JAK2
and STAT5, the activation of ERKs appears to be refractory to
stimulation with rhPRL. Consistent results were obtained in four
independent studies using the same cell lines (data not shown).

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 7.
Constitutive activation of MAP kinase in
unstimulated 32D 178 cells. 32Dcl3 cells
(lanes 1-3 and 10-12), 32D 178
clone 2 cells (lanes 4-6 and 13-15),
and 32D/hPRLR clone 1 cells (lanes 7-9 and
16-18) were stimulated with either 100 units/ml mIL-3
(lanes 1-9) or 1.0 µg/ml rhPRL
(lanes 10-18). Cells were stimulated for 0, 5, or 15 min prior to lysis, and the time of stimulation is indicated at
the top of each lane. Fifty micrograms of total
cellular protein were run on an 8% polyacrylamide gel. The proteins
were transferred to an Immobilon membrane, and the filter was
immunoblotted with either anti-ACTIVE antibody, which specifically
recognizes the activated form of ERK2 and ERK1 (top), or
with a polyclonal anti-MAP kinase antibody that recognizes both ERK1
and ERK2. The positions of ERK1 and ERK2 are indicated by the
arrows on the right in each panel.
Lane numbers are indicated at the bottom of each
lane.
|
|
Phosphorylation of Specific Substrates in 32D
178
Cells--
Numerous proteins have been reported to become
phosphorylated on tyrosine residues following stimulation of cells with
PRL. These include an adapter protein known as SHC (44). We have examined the phosphorylation of SHC in 32D
178 cells to determine whether the presence of the activated PRLR resulted in its constitutive phosphorylation. SHC was immunoprecipitated from all three cell lines
and immunoblotted with an anti-phosphotyrosine monoclonal antibody
(Fig. 8). Although three forms of SHC are
known to exist, only two forms were observed to become phosphorylated
following IL-3 stimulation of either 32Dcl3 or 32D/hPRLR cells (Fig. 8, lanes 1-3 and 7-9). We suspect that
these may represent the 52- and 46-kDa forms of SHC, although they do
not migrate at their reported molecular weights. Both of these forms of
SHC were also phosphorylated in unstimulated 32D
178 cells, and
stimulation with mIL-3 increased their extent of phosphorylation (Fig.
8, lanes 4-6). Stimulation of 32Dcl3 cells with
rhPRL did not result in the appearance of tyrosine-phosphorylated SHC
(data not shown). Stimulation of 32D/hPRLR cells with rhPRL resulted in
the increased phosphorylation of both forms of SHC (Fig. 8,
lanes 14 and 15); however, compared
with the studies with mIL-3, rhPRL does not induce as robust a
phosphorylation of SHC. A longer time of exposure was required to
detect the phosphorylation of SHC induced by rhPRL. This longer
exposure time can be appreciated by comparing the apparent amount of
phosphorylated SHC present in the 32D
178 cells in both panels of
Fig. 8 (compare lanes 4 and 10). The
constitutive phosphorylation of two forms of SHC was observed in
unstimulated 32D
178 cells, and stimulation with rhPRL did not result
in a dramatic increase in the amount of phosphorylated SHC. A doublet of tyrosine-phosphorylated proteins in the 140-150-kDa range was observed to co-immunoprecipitate with SHC in cells stimulated with
mIL-3 (Fig. 8, lanes 2, 3,
5, 6, 8, and 9). Although
this protein is not detected in lanes 10-15 of
Fig. 8, longer exposures revealed the presence of this phosphoprotein.
We suspect that this protein represents SHIP, the SH2-containing
phosphatidylinositol 5-phosphatase that co-immunoprecipitates with SHC
(34, 35, 45).

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 8.
Constitutive phosphorylation of SHC in
unstimulated 32D 178 cells. 32Dcl3 cells
(lanes 1-3), 32D 178 clone 2 cells
(lanes 4-6 and 10-12), and 32D/hPRLR
clone 1 cells (lanes 7-9 and 13-15)
were stimulated with either 100 units/ml mIL-3 (lanes
1-9) or 0.5 µg/ml rhPRL (lanes
10-15). Cells were stimulated for 0, 5, or 15 min prior to
lysis, and the time of stimulation is indicated at the top
of each lane. The lysates were immunoprecipitated with a
polyclonal antibody to SHC, and the immunoprecipitated proteins were
resolved on an 8% polyacrylamide gel. The resolved proteins were
transferred to an Immobilon membrane and immunoblotted with
antiphosphotyrosine monoclonal antibody 4G10. The positions of
molecular mass markers are indicated on the left, and the
positions of SHC are indicated on the right. The immunoblot
was reprobed with a monoclonal antibody to SHC, revealing equal amounts
of SHC in each of the different cell lines under each of the conditions
employed in this experiment (data not shown). Lane numbers are
indicated at the bottom of each lane.
|
|
The
178 Deletion Mutant Can Also Induce Growth
Factor-independent Proliferation of the Nb2 Cell Line--
To
demonstrate that the effects of the
178 deletion mutant were not
specific to the 32Dcl3 cell line, similar studies were also conducted
with the PRL-dependent rat pre-T lymphoma cell line, Nb2
(46). The Nb2 cell line was transfected with expression vectors
encoding either the full-length hPRLR or the
178 deletion mutant,
transfected cells obtained by selection for resistance to G418, single
cell-derived clones obtained as described above. Consistent with the
results described above, transfection of the Nb2 cell line with the
178 expression vector resulted in the appearance of a population of
cells that grew in the absence of PRL. As shown in Fig.
9, the proliferation of Nb2 cells was
stimulated by the addition of oPRL, although some proliferation of Nb2
cells was observed when cells were cultured in the medium containing horse serum alone. This apparently reflects the presence of low amounts
of lactogenic hormones in the preparation of horse serum used in these
studies. The Nb2
178 cells proliferated in both the absence and
presence of exogenous oPRL (Fig. 9). Nb2
178 cells did not
proliferate as fast in the absence of additional oPRL as did the cells
cultured in the presence of oPRL (Fig. 9). This is not surprising,
since, unlike the 32Dcl3 cells, Nb2 cells do have an endogenous
PRLR.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 9.
The 178 deletion
mutant induces growth factor-independent proliferation of Nb2
cells. The proliferation of Nb2 and Nb2 cells transfected with the
178 expression vector was examined as described in Fig. 2 above.
Cells were cultured in RPMI 1640 medium supplemented with 10% horse
serum, 1 mM L-glutamine, and antibiotics as
indicated under "Materials and Methods." The number of viable cells
was determined at the indicated times by direct counting of cells that
excluded trypan blue. , Nb2 cells grown in the presence of 200 ng/ml
oPRL; , Nb2 cells grown in the absence of additional oPRL; ,
Nb2 178 clone 4 cells grown in the presence of 200 ng/ml oPRL; ,
Nb2 178 cells grown in the absence of additional oPRL.
|
|
The activation of two different signaling molecules was also examined
in Nb2 cells expressing the
178 deletion mutant, JAK2 and ERKs.
Nb2/hPRLR clone 7 and Nb2
178 clone 4 cells were stimulated with rat
PRL for 0-15 min, cell lysates were prepared, and the phosphorylation
of JAK2 was examined as described in Fig. 5 above. A small but readily
detectable amount of tyrosine-phosphorylated JAK2 was detected in the
unstimulated Nb2
178 clone 4 cells, and the amount of
tyrosine-phosphorylated JAK2 increased following stimulation with rat
PRL (Fig. 10A,
lanes 1-3). In contrast, very little, if any,
tyrosine-phosphorylated JAK2 was detected in unstimulated Nb2/hPRLR
clone 7 clone cells, although tyrosine-phosphorylated JAK2 was easily
detected in these cells following stimulation with rat PRL (Fig.
10A, lanes 4-6). Consistent with the
data shown in Fig. 5, as well as all previous studies on the activation
of JAK2 by PRL (33, 38-40), there was no difference in the amount of
JAK2 in either of the cell lines at any of the time points examined
(data not shown).

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 10.
Activated JAK2 and MAP kinase are present in
Nb2 cells transfected with the 178 deletion
mutant. The Nb2 178 clone 4 and Nb2/hPRLR clone 7 cells were
cultured overnight in RPMI medium without any serum to reduce the level
of activated signaling molecules. A, the cells were
stimulated for 0-15 min with 10 nM rat PRL. Cell lysates
were prepared, and JAK2 protein was immunoprecipitated and resolved on
a 8% SDS-polyacrylamide gel. The immunoblot was probed with
anti-phosphotyrosine antibody. The position of JAK2 was determined by
reprobing the blot with anti-JAK2 antibody. The amount of JAK2 in each
lane was identical (data not shown). B, the cells were
stimulated with 100 nM oPRL for 0-15 min, the cells were
pelleted, and whole cell lysates were prepared. Twenty-five micrograms
of each protein were resolved on a 8% SDS-polyacrylamide gel, and the
immunoblot was probed with anti-ACTIVE MAP kinase antibody.
C, the immunoblot shown in B was reprobed with
anti-MAP kinase antibody that reacted with both ERK1 and ERK2. The
positions of ERK1 and ERK2 are indicated on the right in
B and C. The time of stimulation with PRL (in
minutes) is indicated at the top of each lane.
Lanes numbers are indicated at the bottom of each
lane.
|
|
The amount of activated ERK1 and ERK2 was also examined in both
Nb2
178 clone 4 and Nb2/hPRLR clone 7 cells using the same approach
utilized in Fig. 7. Both cell lines were stimulated with 100 nM oPRL for 0-15 min, whole cell lysates were prepared,
and immunoblots were analyzed with either anti-ACTIVE MAP kinase
antibody or anti-MAP kinase antibody. As shown in Fig. 10B,
a low amount of activated ERK1 and ERK2 was present in unstimulated
Nb2
178 clone 4 cells, and the amount of activated ERK did not
increase following stimulation with oPRL (Fig. 10B,
lanes 10-12). In contrast, no activated ERK1 or
ERK2 was detected in unstimulated wild type Nb2 cells or Nb2/hPRLR
clone 7 cells; however, there was a dramatic increase in the amount of
activated ERK following PRL stimulation, as indicated by
immunoblotting. There was no change in the amount of either ERK1 or
ERK2 in any of the cell lines at any of the time points examined (Fig.
10C). These data are consistent with the results obtained
with the 32D
178 cells examined in Fig. 7. In both studies there was
a low level of activated ERK present in cells expressing the
178
deletion mutant, and stimulation with either oPRL or rhPRL did not
stimulate an increase in the amount of activated ERK.
Expression of the
178 Mutant of the PRLR Suppresses
Apoptosis of 32Dcl3 Cells following Cytokine Withdrawal--
As
noted above, the proliferation and viability of 32Dcl3 cells is
critically dependent upon stimulation with exogenous IL-3. Cultivation
of these cells in the absence of growth factors results in their death
within 48-60 h. Numerous oncogenes are able to render 32Dcl3 cells
growth factor-independent with a concomitant suppression of apoptosis
(47-52). We were interested in determining whether expression of the
178 deletion mutant of the PRLR suppressed apoptosis. Both 32Dcl3
and 32D
178 cells were cultured in the presence or absence of 10%
WEHI-3 conditioned media for 0-72 h. DNA was isolated from these cells
at various time points, and the induction of DNA fragmentation was
assessed by agarose gel electrophoresis. DNA fragmentation was detected
in 32Dcl3 cells cultured in the absence of mIL-3 by 16 h, and the
200-base pair ladder was evident at 36 h (Fig.
11, lanes 1-7).
Cultivation of 32Dcl3 cells in mIL-3 did not result in DNA
fragmentation (Fig. 11, lanes 8-11). No DNA
fragmentation was observed in the 32D
178 clone 2 cells, regardless
of whether they were cultured in the presence or absence of mIL-3 (Fig.
11, lanes 12-22). These results indicate that in
addition to being growth factor-independent, cells expressing the
178 deletion mutant of the hPRLR do not undergo apoptosis following
growth factor withdrawal.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 11.
Lack of apoptosis in
32D 178 cells cultured in the absence of
mIL-3. The 32Dcl3 and 32D 178 clone 2 cells were cultured in the
presence or absence of mIL-3 for 0-72 h. At the indicated times, DNA
was isolated from these cells cultured under both conditions. The
presence of DNA fragmentation, indicative of apoptosis, was examined by
electrophoresis of 10 µg of each DNA sample on a 1.5% agarose gel.
Analysis of the 32Dcl3 cell line is shown in the top
panel, while analysis of the 32D 178 clone 2 cell line is
shown in the bottom panel. The time of DNA
isolation is indicated at the top of each lane.
Lanes marked M contain molecular size markers.
The lane numbers are indicated at the bottom of each
lane.
|
|
 |
DISCUSSION |
The binding of PRL to its receptor activates a specific series of
signaling events within the cell. The critical initial event appears to
be the ligand-induced dimerization of the receptor leading to the
activation of specific tyrosine kinases (53, 54). These kinases include
both the JAK2 tyrosine kinase (33, 38-40) and the
src-related tyrosine kinase Fyn (55). Activation of JAK2
leads to the phosphorylation and activation of the transcription factor
STAT5 (56, 57). STAT5, also known as mammary gland factor, is required
for the induction of specific transcription products such as
-casein
(56-58). Other signaling pathways that are activated include Ras, Raf,
and MAP kinase (42, 44, 59) as well as phosphatidylinositol 3-kinase
(60). The means by which these other signaling pathways are regulated
has not been demonstrated; however, it is possible that they may lie
downstream of src-like tyrosine kinases. Numerous other
proteins become tyrosine-phosphorylated following activation of the
PRLR, including the receptor itself (33, 38-40), the SH2-containing
adapter protein SHC (44), and the insulin receptor substrate-1 (61).
With the exception of insulin receptor substrate-1, these proteins and
enzymes appear to be critical in regulating the response of all cells
to growth factors and cytokines.
In this report, we have demonstrated that deletion of the extracellular
ligand binding domain of the human PRLR results in the constitutive
activation of the receptor. Evidence supporting this conclusion
includes the following: 1) expression of this truncated receptor leads
to growth factor-independent proliferation of a growth
factor-dependent myeloid cell line; 2) an elevated level of
tyrosine-phosphorylated proteins was observed in unstimulated 32D
178
cells; 3) constitutive activation of both JAK2 and STAT5 was observed
in unstimulated 32D
178 cells; 4) ERKs were constitutively activated
in the absence of IL-3 or PRL stimulation; 5) the SH2-containing adapter protein SHC was phosphorylated in unstimulated 32D
178 cells;
and 6) in contrast to 32Dcl3 cells, which undergo apoptosis upon IL-3
withdrawal, 32D
178 cells did not undergo apoptosis under the same
conditions. Similar data were obtained when the
178 deletion mutant
was expressed in the Nb2 cell line, indicating that these results are
not cell type-specific. These data clearly support the conclusion that
deletion of the majority of the extracellular domain of the PRLR
results in the constitutive activation of this receptor. The majority,
if not all, of the proteins involved in signal transduction by the
PRLR, also appear to be activated/phosphorylated by the
178 deletion
mutant. To date we have not observed the phosphorylation of any novel
proteins that have not been observed to be phosphorylated following the
binding of PRL to its receptor. This suggests that the constitutively
active mutant of the PRLR utilizes the same normal signaling pathways
used by the PRLR. Although identical results were obtained in the
analysis of five independent clones of 32D/hPRLR cells, five different
clones of 32Dcl3 cells expressing the FLAG-tagged hPRLR, five different clones of 32D
178 cells, and five independent clones of cells expressing the FLAG-tagged
178 protein, we cannot rule out the possibility that additional genetic changes have occurred to these cells during the selection of these cells. Secondary genetic changes in
the 32Dcl3 cells expressing the hPRLR cannot be readily identified, and
for them to be present in all of the independently derived clones would
suggest that there must be a tremendous genetic pressure for them to
occur. DNA sequence analysis would be required to determine whether
additional mutations have occurred in the
178 protein; DNA sequence
analysis is beyond the scope of the present investigation but will be
examined in the future.
Other investigators have previously described the constitutive
activation of the rabbit PRLR following deletion of amino acids 103-203. The biological assay utilized by these authors was the transcriptional activation of a reporter gene construct in which the
promoter of the
-lactoglobulin gene was fused to the
chloroamphenicol acetyltransferase reporter gene (22). This
constitutively activated receptor has also been shown to induce
expression of
-casein in HC11 in the absence of PRL stimulation
(22). The ability of this mutant receptor to induce transcription of a
gene known to be induced by PRL, in a growth factor-independent manner,
demonstrates that this deletion mutant of the PRLR has acquired some of
the properties of an activated growth factor receptor. These authors did not examine the ability of their activated receptor mutant to
activate downstream signaling molecules such as JAK2, STAT5, ERKs, or
any other signaling proteins known to lie downstream of the PRLR (22).
Furthermore, they did not report the ability of their activated
receptor to induce growth factor-independent proliferation of
PRL-dependent cell lines (22).
Gourdou et al. (22) have reported that the deletion of amino
acids 103-203 (domain S2) resulted in the constitutive activation of
the PRLR; however, the deletion of amino acids 3-103 (domain S1) or
3-203 (S1,S2) did not result in the activation of the receptor. Based
upon these data, Gourdou et al. have suggested that
sequences in the S1 region can activate the PRLR and that sequences in
the S2 region function to suppress the activity of S1 (22). We do not
believe this hypothesis of Gourdou et al. to be correct,
since the
178 we have generated is very similar to the S1,S2
deletion used in their studies (see below). As noted above, our
strategy in making the
178 deletion mutant was to delete as much of
the extracellular ligand binding region as possible and to leave the WSXWS motif intact. The major difference between our
178
deletion mutant and the inactive 3-203 deletion mutant made by Gourdou et al. is that our deletion mutant contains the
WSXWS sequence, while their deletion mutant lacks this
conserved sequence (Fig. 1). The three-dimensional structure of the
human PRLR bound to human growth hormone indicates that the
extracellular domain of the receptor forms two
-sheet barrels (62).
The WSXWS sequence is thought to be critical in maintaining
the structure of the
-sheet barrel adjacent to the plasma membrane
(62). We hypothesize that this WSXWS sequence is absolutely
critical for the constitutive activation of our
178 mutant. Our
model predicts that dimerization of receptors containing deletions of
the extracellular ligand binding domains is driven by the hydrophobic
interaction between the WSXWS sequence on one receptor
molecule with the same sequence on a second receptor molecule. Such
interactions would remove four tryptophan side chains from water and
provide sufficient energy to make this a stable interaction. Since the
tryptophan residues of the WSXWS sequence are normally
buried, these side chains normally would not contribute to the
dimerization of receptor monomers. This model would predict that these
tryptophan residues could be mutated to other hydrophobic amino acids,
such as phenylalanine. Replacement of the tryptophans with charged
amino acids, however, should render these constitutively activated
receptors nonfunctional. This hypothesis will be directly tested by
mutation of the amino acids predicted to be critical in the
ligand-independent receptor dimerization. We do not believe that the S2
region functions as a repressor of the S1 region as suggested by Goudou
et al. (22), since our mutant essentially deletes both S1
and S2.
Although it is clear that the PRLR is able to induce proliferation of
the Nb2 rat pre-T lymphoma cell line as well as induce proliferation of
factor-dependent myeloid cell lines such as those utilized
in this study, it is not clear whether the PRLR can stimulate proliferation of other cells, such as normal mammary epithelial cells.
Although other investigators have demonstrated that PRL can stimulate
the proliferation of a human breast cancer cell line (63, 64), it is
not clear whether PRL alone can stimulate the proliferation of mammary
epithelial cells in the mammary gland. Both PRL and its receptor play a
critical role in the development of the mammary gland and the induction
of lactation (65, 66). Due to the complexity of growth factors and
steroid hormones that influence the development of the mammary gland,
it is not clear whether PRL alone can stimulate proliferation of cells
in the mammary gland. Analysis of female mice carrying homozygous
deletion of either the PRLR or STAT5a clearly demonstrate that the PRLR signal transduction is critical in the development of the mammary gland
(65, 67); however, analysis of these mice does not answer the question
of whether PRL and its receptor can stimulate proliferation of cells in
this tissue. We believe that we have established a system that will
allow exploration of this question.
The data we have presented clearly indicate that the
178 deletion
mutant is able to induce growth factor-independent proliferation of
factor-dependent hematopoietic cells as well as induce
constitutive activation of several signaling molecules that lie
downstream of the PRLR. Furthermore, factor-dependent cells
expressing this constitutively activated version of the PRLR do not
undergo apoptosis when growth factor is withdrawn. These properties are
among those one would expect of an activated oncogene; however, we have
not demonstrated that the
178 deletion mutant is able to transform either fibroblasts or mammary epithelial cells. Future studies will
address the oncogenic potential of this constitutively active mutant of
the PRLR.
 |
ACKNOWLEDGEMENTS |
We acknowledge the services of the University
of Colorado Cancer Center DNA Sequencing Core Facility in support of
this research. We thank Elizabeth Burton for discussions on this
project and Dr. Arthur Gutierrez-Hartmann for comments on this
manuscript. We also thank Dr. Paul Kelly for providing the human PRLR
cDNA and Dr. Andrew Larner for providing the anti-STAT5 antibody.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
(NIH) Grant DK48879 and a University of Colorado Cancer Center/Wines for Life seed grant. This investigation also made use of the University of Colorado Cancer Center DNA Sequencing Core Facility which is supported by NIH Grant CA46934.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Member of the Medical Scientist Training Program, which is
supported by NIH Grant GM08497.
To whom correspondence should be addressed: Dept. of
Pathology, Box B-216, University of Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80262. Tel.: 303-315-4787; Fax:
303-315-6721; E-mail: steve.anderson{at}uchsc.edu.
2
S. M. Anderson, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
STAT, signal
transducer and activator of transcription;
STAT5, signal transducer and
activator of transcription-5;
GAPDH, glyceraldehyde-3-phosphate
dehydrogenase;
ERK, extracellular signal-regulated kinase;
ERK1 and
ERK2, extracellular signal-regulated kinase 1 and 2, respectively;
PRL, prolactin;
PRLR, prolactin receptor;
hPRL, human prolactin;
oPRL, ovine
prolactin;
hPRLR, human prolactin receptor;
rPRL, rat prolactin;
rhPRL, recombinant human prolactin;
IL, interleukin;
mIL, murine interleukin;
JAK2, Janus kinase 2;
MAP, mitogen-activated protein.
 |
REFERENCES |
-
Darnell, J. E.,
Kerr, I. M.,
and Stark, G. R.
(1994)
Science
264,
1415-1421[Medline]
[Order article via Infotrieve]
-
Downward, J.,
Yarden, Y.,
Mayes, G.,
Scrace, G.,
Totty, N.,
Stockwell, P.,
Ullrich, A.,
Schlessinger, J.,
and Waterfield, M. D.
(1984)
Nature
307,
521-527[Medline]
[Order article via Infotrieve]
-
Sherr, C. J.,
Rettenmier, C. W.,
Sacca, R.,
Roussel, M. F.,
Look, A. T.,
and Stanley, E. R.
(1985)
Cell
41,
665-676[Medline]
[Order article via Infotrieve]
-
Williams, D. E.,
Eisenman, J.,
Baird, A.,
Rauch, C.,
Van Ness, K.,
March, C. J.,
Park, L. S.,
Martin, U.,
Mochizuki, D. Y.,
Boswell, H. S.,
Burgess, G. S.,
Cosman, D.,
and Lyman, S. D.
(1990)
Cell
63,
167-174[Medline]
[Order article via Infotrieve]
-
Huang, E.,
Nocka, K.,
Beier, D. R.,
Chu, T.,
Buck, J.,
Lahm, H.,
Wellner, D.,
Leder, P.,
and Bessmer, P.
(1990)
Cell
63,
225-233[Medline]
[Order article via Infotrieve]
-
Zsebo, K. M.,
Williams, D. A.,
Geissler, E. N.,
Broudy, V. C.,
Martin, F. N.,
Atkins, H. L.,
Hsu, R.,
Birkett, N. C.,
Okino, K. H.,
Murdock, D. C.,
Jacobsen, F. W.,
Langlely, K. E.,
Smith, K. A.,
Takeishi, T.,
Cattanach, B. M.,
Galli, S. J.,
and Suggs, S. V.
(1990)
Cell
63,
213-224[Medline]
[Order article via Infotrieve]
-
Gonda, T. J.,
and D'Andrea, R. J.
(1997)
Blood
89,
355-369[Free Full Text]
-
Roussel, M. F.,
Downs, C. P.,
Rettenmier, C. W.,
and Sherr, C. J.
(1988)
Cell
55,
979-988[Medline]
[Order article via Infotrieve]
-
Woolford, J.,
McAuliffe, A.,
and Rohrschneider, L. R.
(1988)
Cell
55,
965-977[Medline]
[Order article via Infotrieve]
-
Lax, I.,
Kris, R.,
Ullrich, A.,
Hayman, M. J.,
Beug, H.,
and Schlessinger, J.
(1985)
EMBO J.
4,
3179-3182[Abstract]
-
Bargmann, C. I.,
and Weinberg, R. A.
(1988)
EMBO J.
7,
2043-2052[Abstract]
-
Bargmann, C. I.,
Hung, M.-C.,
and Weinberg, R. A.
(1986)
Cell
45,
649-657[Medline]
[Order article via Infotrieve]
-
Wendling, F.,
Varlet, P.,
Charon, M.,
and Tambourin, P.
(1986)
Virology
149,
242-246[Medline]
[Order article via Infotrieve]
-
Souyri, M.,
Vigon, I.,
Penciolelli, J.,
Heard, J.,
Tambourin, P.,
and Wendling, F.
(1990)
Cell
63,
1137-1147[Medline]
[Order article via Infotrieve]
-
Vigon, I.,
Mornon, J.,
Cocault, L.,
Mitjavila, M.,
Tambourin, P.,
Gisselbrecht, S.,
and Souyri, M.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
5640-5644[Abstract]
-
Longmore, G. D.,
and Lodish, H. F.
(1991)
Cell
67,
1089-1102[Medline]
[Order article via Infotrieve]
-
D'Andrea, R.,
Raynen, J.,
Moretti, P.,
Lopez, A.,
Goodall, G. J.,
Gonda, T. J.,
and Vadas, M.
(1994)
Blood
83,
2802-2808[Abstract/Free Full Text]
-
Jenkins, B. J.,
D'Andrea, R. J.,
and Gonda, T. J.
(1995)
EMBO J.
14,
4276-4287[Abstract]
-
Hannemann, J.,
Hara, T.,
Kawai, M.,
Miyajima, A.,
Ostertag, W.,
and Stocking, C.
(1995)
Mol. Cell. Biol.
15,
2402-2412[Abstract]
-
Jenkins, B. J.,
Bagley, C. J.,
Woodcock, J.,
Lopez, A. F.,
and Gonda, T. J.
(1996)
J. Biol. Chem.
271,
29707-29714[Abstract/Free Full Text]
-
D'Andrea, R. J.,
Barry, S. C.,
Moretti, P. A. B.,
Jones, K.,
Ellis, S.,
Vadas, M. A.,
and Goodall, G. J.
(1996)
Blood
87,
2641-2648[Abstract/Free Full Text]
-
Gourdou, I.,
Gabou, L.,
Paly, J.,
Kermabon, A. Y.,
Belair, L.,
and Djiane, J.
(1996)
Mol. Endocrinol.
10,
45-56[Abstract]
-
Greenberger, J. S.,
Sakakeeny, M. A.,
Humphries, K. C.,
Eaves, C. J.,
and Eckner, R. J.
(1983)
Proc. Natl. Acad. Sci. U. S. A.
80,
2931-2935[Abstract]
-
Anderson, S. M.,
and Jorgensen, B.
(1995)
J. Immunol.
155,
1660-1670[Abstract]
-
Hunter, S.,
Koch, B. L.,
and Anderson, S. M.
(1997)
Mol. Endocrinol.
11,
1213-1222[Abstract/Free Full Text]
-
Anderson, S. M.,
Burton, E. A.,
and Koch, B. L.
(1997)
J. Biol. Chem.
272,
739-745[Abstract/Free Full Text]
-
Burton, E. A.,
Hunter, S.,
Wu, S. C.,
and Anderson, S. M.
(1997)
J. Biol. Chem.
272,
16189-16195[Abstract/Free Full Text]
-
Metcalf, D.
(1985)
Blood
65,
357-362[Abstract]
-
Rui, H.,
Djeu, J. Y.,
Evans, G. A.,
Kelly, P. A.,
and Farrar, W. L.
(1992)
J. Biol. Chem.
267,
24076-24081[Abstract/Free Full Text]
-
Isfort, R. J.,
Stevens, D.,
May, W. S.,
and Ihle, J. N.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
7982-7986[Abstract]
-
Isfort, R. J.,
Huhn, R. D.,
Frackelton, A. R.,
and Ihle, J. N.
(1988)
J. Biol. Chem.
263,
19203-19209[Abstract/Free Full Text]
-
Linnekin, D.,
and Farrar, W.
(1990)
Biochem. J.
27,
317-324
-
Silvennoinen, O.,
Witthuhn, B. A.,
Quelle, F. W.,
Cleveland, J. L.,
Yi, T.,
and Ihle, J. N.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
8429-8433[Abstract/Free Full Text]
-
Lioubin, M. N.,
Algate, P. A.,
Tsai, S.,
Carlberg, K.,
Aebersold, R.,
and Rohrschneider, L. R.
(1996)
Genes Dev.
10,
1084-1095[Abstract]
-
Ware, M. D.,
Rosten, P.,
Damen, J. E.,
Liu, L.,
Humphrey, P. A.,
and Krystal, G.
(1996)
Blood
88,
2833-2840[Abstract/Free Full Text]
-
Cutler, R. L.,
Liu, L.,
Damen, J. E.,
and Krystal, G.
(1993)
J. Biol. Chem.
268,
21463-21465[Abstract/Free Full Text]
-
Okuda, K.,
Sanghera, J. S.,
Pelech, S. L.,
Kanakura, Y.,
Hallek, M.,
Griffin, J. D.,
and Druker, B. J.
(1992)
Blood
79,
2880-2887[Abstract]
-
Rui, H.,
Kirken, R. A.,
and Farrar, W. L.
(1994)
J. Biol. Chem.
269,
5364-5368[Abstract/Free Full Text]
-
Lebrun, J.-J.,
Ali, S.,
Sofer, L.,
Ullrich, A.,
and Kelly, P. A.
(1994)
J. Biol. Chem.
269,
14021-14026[Abstract/Free Full Text]
-
David, M.,
Petricoin, E. F., III,
Igarashi, K.-I.,
Feldman, G. M.,
Finbloom, D. S.,
and Larner, A. C.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
7174-7178[Abstract]
-
Buckley, A. R.,
Rao, Y.-P.,
Buckley, D. J.,
and Gout, P. W.
(1994)
Biochim. Biophys. Acta
204,
1158-1164
-
Piccoletti, R.,
Maroni, P.,
Bendinelli, P.,
and Bernelli-Zazzera, A.
(1994)
Biochem. J.
303,
429-433[Medline]
[Order article via Infotrieve]
-
Boulton, T. G.,
Nye, S. H.,
Robbins, D. J.,
Ip, N.,
Radziejewska, E.,
Morgenbesser, S. D.,
DePinho, R. A.,
Panayotatos, N.,
Cobb, M. H.,
and Yancopoulos, G. D.
(1991)
Cell
65,
663-675[Medline]
[Order article via Infotrieve]
-
Erwin, R. A.,
Kirken, R. A.,
Malabarba, M. G.,
Farrar, W. L.,
and Rui, H.
(1996)
Endocrinology
136,
3512-3518[Abstract]
-
Damen, J. E.,
Liu, L.,
Rosten, P.,
Humphries, R. K.,
Jefferson, A. B.,
Majerus, P. W.,
and Krystal, G.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
1689-1693[Abstract/Free Full Text]
-
Shiu, R. P. C.,
Elsholtz, H. P.,
Tanaka, T.,
Friesen, H. G.,
Gout, P. W.,
Beer, C. T.,
and Noble, R. L.
(1983)
Endocrinology
113,
159-165[Abstract]
-
Anderson, S. M.,
Carroll, P. M.,
and Lee, F. D.
(1989)
Oncogene
5,
317-325
-
Rovera, G.,
Valtieri, M.,
Mavilio, F.,
and Reddy, E. P.
(1987)
Oncogene
1,
29-35[Medline]
[Order article via Infotrieve]
-
Metcalf, D.,
Roberts, T. M.,
Cherington, V.,
and Dunn, A. R.
(1987)
EMBO J.
6,
3703-3709[Abstract]
-
Cleveland, J. L.,
Dean, M.,
Rosenberg, N.,
Wang, J. Y. J.,
and Rapp, U. R.
(1989)
Mol. Cell. Biol.
9,
5685-5695[Medline]
[Order article via Infotrieve]
-
Askew, D. S.,
Ashmun, R. A.,
Simmons, B. C.,
and Cleveland, J. L.
(1991)
Oncogene
6,
1915-1922[Medline]
[Order article via Infotrieve]
-
Cleveland, J. L.,
Troppmair, J.,
Packham, G.,
Askew, D. S.,
Lloyd, P.,
Gonzalez-Garcia, M.,
Nunez, G.,
Ihle, J. N.,
and Rapp, U. R.
(1994)
Oncogene
9,
2217-2226[Medline]
[Order article via Infotrieve]
-
Wells, J. A.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
1-6[Abstract/Free Full Text]
-
Goffin, V.,
and Kelly, P. A.
(1997)
J. Mammary Gland Biol. Neoplasia
2,
7-17[Medline]
[Order article via Infotrieve]
-
Clevenger, C. V.,
and Medaglia, M. V.
(1994)
Mol. Endocrinol.
8,
674-681[Abstract]
-
Wakao, H.,
Gouilleux, F.,
and Groner, B.
(1994)
EMBO J.
13,
2182-2191[Abstract]
-
Gouilleux, F.,
Wakao, H.,
Mundt, V.,
and Groner, B.
(1994)
EMBO J.
13,
4361-4369[Abstract]
-
Liu, X.,
Robinson, G. W.,
Gouilleux, F.,
Groner, B.,
and Hennighausen, L.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
8831-8835[Abstract]
-
Carey, G. B.,
and Liberti, J. P.
(1995)
Arch. Biochem. Biophys.
316,
179-189[CrossRef][Medline]
[Order article via Infotrieve]
-
Al-Sakkaf, K. A.,
Dobson, P. R. M.,
and Brown, B. L.
(1996)
Biochem. Biophys. Res. Commun.
221,
779-784[CrossRef][Medline]
[Order article via Infotrieve]
-
Berlanga, J. J.,
Gualillo, O.,
Buteau, H.,
Applanat, M.,
Kelly, P. A.,
and Edery, M.
(1997)
J. Biol. Chem.
272,
2050-2052[Abstract/Free Full Text]
-
Sommers, W.,
Ultsch, M.,
de Vos, A. M.,
and Kossiakoff, A. A.
(1994)
Nature
372,
478-481[CrossRef][Medline]
[Order article via Infotrieve]
-
Biswas, R.,
and Vonderhaar, B. K.
(1987)
Cancer Res.
47,
3509-3514[Abstract]
-
Ginsberg, E.,
and Vonderhaar, B. K.
(1995)
Cancer Res.
55,
2591-2595[Abstract]
-
Ormandy, C. J.,
Camus, A.,
Barra, J.,
Damotte, D.,
Lucas, B.,
Buteau, H.,
Edery, M.,
Brousse, N.,
Babinet, C.,
Binart, N.,
and Kelly, P. A.
(1997)
Genes Dev.
11,
167-178[Abstract]
-
Bole-Feysot, C.,
Goffin, V.,
Edery, M.,
Binart, N.,
and Kelly, P. A.
(1998)
Endocr. Rev.
19,
225-268[Abstract/Free Full Text]
-
Liu, X.,
Robinson, G. W.,
Wagner, K.-U.,
Garrett, L.,
Wynshaw-Boris, A.,
and Hennighausen, L.
(1997)
Genes Dev.
11,
179-186[Abstract]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.