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J Biol Chem, Vol. 274, Issue 50, 35461-35468, December 10, 1999
From the Department of Pathology and Laboratory Medicine,
University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 19104
Prolactin-dependent signaling occurs
as the result of ligand-induced dimerization of the prolactin receptor
(PRLr). While three PRLr isoforms have been characterized in the rat,
studies have suggested the existence of several human isoforms in
breast carcinoma species and normal tissues. Reverse transcription
polymerase chain reaction was performed on mRNA isolated from the
breast carcinoma cell line T47D, revealing two predominant receptor
isoforms: the previously described long PRLr and a novel human
intermediate PRLr. The nucleotide sequence of the intermediate isoform
was found to be identical to the long isoform except for a 573-base pair deletion occurring at a consensus splice site, resulting in a
frameshift and truncated intracytoplasmic domain. Scatchard analysis of
the intermediate PRLr revealed an affinity for PRL comparable with the
long PRLr. While Ba/F3 transfectants expressing the long PRLr
proliferated in response to PRL, intermediate PRLr transfectants
exhibited modest incorporation of [3H]thymidine.
Significantly, however, both the long and intermediate PRLr were
equivalent in their inhibition of apoptosis of the Ba/F3 transfectants
after PRL treatment. The activation of proximal signaling molecules
also differed between isoforms. Upon ligand binding, Jak2 and Fyn were
activated in CHO-K1 cells transiently transfected with the long PRLr.
In contrast, the intermediate PRLr transfectants showed equivalent
levels of Jak2 activation but only minimal activation of Fyn. Last,
Northern analysis revealed variable tissue expression of intermediate
PRLr transcript that differed from that of the long PRLr. Taken
together, differences in signaling and tissue expression suggest that
the human intermediate PRLr differs from the long PRLr in physiological function.
The neuroendocrine hormone prolactin
(PRL)1 exhibits high homology
to growth hormone and is also related to the peptide hormones of the
interleukin family (1, 2). PRL has been implicated in the proliferation
and differentiation of lobular units as well as the initiation and
maintenance of lactation (3, 4). It has also been shown to be an
essential component of the T cell immune response, serving as a
cofactor for T lymphocyte activation (5, 6). Regulation by PRL may also
extend to the autocrine level as the synthesis and secretion of PRL by
mitogen-stimulated T cells (7, 8) and within breast epithelium (9, 10) has been identified.
PRL exerts its effects at the molecular level by inducing the
homodimerization of the prolactin receptor (PRLr). A member of the
cytokine receptor family, the PRLr lacks intrinsic enzymatic activity,
thus requiring the activation of associated kinases and other signaling
factors for ligand-driven transduction. Two protein-tyrosine kinases
found in association with the PRLr are p59fyn (11) and
p120jak2 (12-15). Through Jak2, PRL stimulation activates Stat
family members in lymphocytes (16) and breast tissues (17, 18),
resulting in the initiation of transcription for interferon regulatory
factor-1 and While the diversity of PRL function is in part mediated by a variety of
signaling cascades, differences in function may also be attributed to
the wide variety of PRLr forms observed in nature. As members of the
cytokine receptor superfamily, the PRLr isoforms show significant
sequence similarity in their extracellular ligand-binding domains.
Within the membrane-proximal region of the intracytoplasmic domain of
PRL receptors and other superfamily members lie the conserved Box 1 and
Box 2 motifs. Box 1 is a hydrophobic, proline-rich region that
resembles an SH3 binding domain (22-24). The Box 2 domain is
hydrophobic and acidic, and its signaling function is largely
uncharacterized. Several isoforms of the PRLr have been identified in
both mammals (25-31) and birds (32-34). The most well characterized
isoforms are those found in the rat: the short form (45 kDa) (35), long
form (80-85 kDa) (36), and a mutant intermediate form found on the
PRL-dependent rat T cell lymphoma line Nb2 (65 kDa) (Fig.
3B) (36, 37). In humans, the only PRLr isoform characterized
thus far is the long form cloned from the liver (Fig. 3A)
(38). Previous studies have, however, provided evidence that other
human PRLr isoforms may be expressed in human tissues (30, 39).
In this study, we identify a novel isoform of the human PRLr cloned
from the human breast cancer cell line T47D. The isoform is analyzed
for 1) in vivo surface expression and its ability to bind
ligand, 2) induction of cell proliferation and cell survival in
response to ligand, 3) the ability to activate associated kinases, and
4) the relative levels of its corresponding mRNA in normal human tissues.
mRNA Isolation and Reverse Transcription PCR--
T47D
cells, an estrogen receptor/PRLr-positive human breast cancer cell
line, were used for mRNA isolation. Whole RNA was purified from
107 washed cells using Trizol reagent (Life Technologies,
Inc.) as described previously (39). Messenger RNA was then purified
from the whole RNA preparation with oligo(dT)-cellulose (Invitrogen, San Diego, CA). 5 µg of T47D mRNA was used for first strand
synthesis of cDNA using the Superscript II RT cDNA kit (Life
Technologies, Inc.). Negative controls consisted of reactions
containing no T47D mRNA or no reverse transcriptase. A positive
control reaction consisted of chloramphenicol acetyltransferase
mRNA template. For polymerase chain reaction, 2 µl of the
corresponding cDNA reactions were added to a 50-µl reaction
containing 5-µl 10× PCR buffer, 3 µl of 25 mM
MgCl2, 1 µl of 10 mM dNTP mix, 5 units of Taq polymerase (Life Technologies, Inc.), and primers for
amplification. As the positive control reaction, primers A
(5'-GACATGGAAGCCATCACAGAC-3') and B (5'-CGACCGTTCAGCTGGATATTA-3') were
used to amplify a fragment of the chloramphenicol acetyltransferase
gene from control cDNA. The PRLr gene amplification reaction
contained primers PRLR-F3 (5'-ATGAAGGAAAATGTGGCA-3') and PRLR-1
(5'-TCAGTGAAAGGAGTGTGT-3'), which correspond to the 5'- and 3'-ends of
the human long PRLr open reading frame. The primary cycle of the
reaction consisted of 94 °C for 2 min, 42 °C for 1 min, 72 °C
for 3 min, and 94 °C for 2 min, which was followed by 30 cycles of
94 °C for 30 s, 47 °C for 30 s, and 72 °C for 2 min.
It was then extended at 72 °C for 3 min. Isolated PCR fragments were
subcloned into the TA vector pCR 2.1 (Invitrogen, San Diego, CA) and
analyzed by dideoxynucleotide sequencing. For eukaryotic expression of
the intermediate isoform, the gene was reamplified by PCR with primers
PRLR-Kl (5'-CGAATTCCACCATGAAGGAAAATGTGGCA-3') and PRLR-599'
(5'-GCGCTCGAGTCAGTGAAAGGAGTGTGTAAA-3'), which contain a 5'
EcoRI restriction site and Kozak initiation sequence and a
3' XhoI restriction site, respectively. Alternative 3'
primers were also utilized to remove the tertiary stop codon from the open reading frames of the isoforms, allowing the addition of a
carboxyl-terminal V5 epitope tag when ligated into vector pEF1-V5/HisA (Invitrogen). The intermediate isoform was reamplified with primers PRLR-Kl and PRLR-INT' (5'-GCGCTCGAGGGAGTCCCGGGCTTC-3'), while the long
isoform was reamplified with PRLR-Kl and PRLR-LONG'
(5'-CGCTCGAGGTGAAAGGAGTGTGTAAA-3'). The DNA fragments were
digested with EcoRI and XhoI and ligated into the
corresponding restriction sites of pcDNA3 and pEF1-V5/HisA. The
clones were subsequently checked for amplification errors by
dideoxynucleotide sequencing.
Cell Culture and Transfection--
T47D cells were maintained in
Dulbecco's modified Eagle's medium (Life Technologies) supplemented
with 10% fetal bovine serum and 1% penicillin/streptomycin. The mouse
interleukin 3 (IL-3)-dependent pro-B cell line Ba/F3 was
maintained in RPMI 1640 medium (Life Technologies) supplemented with
10% fetal bovine serum and 1% penicillin/streptomycin in the presence
of 1 ng/ml IL-3 (PeproTech, Rocky Hill, NJ). Chinese hamster ovary
(CHO-K1) cells were maintained in Ham's F-12 medium (Life
Technologies) supplemented with 10% fetal bovine serum and 1%
penicillin/streptomycin. Ba/F3 cells (107) were transfected
with 50 µg of intermediate or long isoform cDNA clones in
pcDNA3 by exposure to a single voltage pulse (0.6 kV, 25 microfarads for 0.1 s) in a Gene Pulser electroporator (Bio-Rad).
Stable clones were obtained by limiting dilution by selection in 750 µg/ml G418. CHO cells (2 × 105) were transiently
transfected with 2 µg of intermediate or long isoform cDNA clones
in pEF1-V5/HisA in conjunction with 2 µg of human Jak2 (gift of Dr.
Roy Duhe) or murine Fyn (gift of Dr. Paul Stein) cDNA in
pEF1-V5/HisA using Fugene 6 (Roche Molecular Biochemicals) as instructed.
Northern Analysis of PRLr Isoform Transfectants and Isoform
Expression in Various Tissues--
Total RNA from cells was isolated
from T47D cells by extraction with Trizol reagent (Life Technologies)
as described previously (39). 10 µg of total RNA was denatured and
subjected to electrophoresis on a 1% agarose formaldehyde gel and
transferred onto a nylon membrane. A cDNA probe generated from bp
73-702 of the extracellular domain of the human PRLr long isoform was
labeled with [
A master tissue blot of human total mRNA
(CLONTECH) was probed with cDNAs specific for
either the intermediate or long PRLr isoforms. Equal loading of
mRNAs was confirmed by the quantitation of eight distinct
housekeeping genes. The cDNA probe specific for the long isoform
was composed of nucleotides 1037-1347 of the long form open reading
frame (38). This entire region is deleted in the intermediate isoform
open reading frame. The probe for the intermediate isoform spans the
573-bp deletion due to alternative splicing. This corresponds to
nucleotides 910-1054 of the intermediate isoform open reading frame
(or 910-1580 of the long isoform open reading frame (38)).
Hybridization conditions were performed as instructed by
CLONTECH. Under these conditions, no
cross-hybridization was observed between isoforms (data not shown). The
blot was exposed to x-ray film for 4 days, and signal intensities were
obtained using ImageQuaNT densitometry software (Molecular Dynamics,
Inc., Sunnyvale, CA).
Immunoblot Analysis--
CHO cell transfectants were lysed in
Laemmli buffer containing SDS and 2-mercaptoethanol (5). Lysates were
electrophoresed through an 8% SDS-polyacrylamide gel and transferred
to nitrocellulose. Nonspecific binding was blocked with 5% milk in
PBS/Tween 20. Antigen was labeled with 1 µg of horseradish
peroxidase-conjugated anti-V5 antibody (Invitrogen) per ml.
Antigen-antibody complexes were visualized by enhanced
chemiluminescence (Amersham Pharmacia Biotech).
Immunofluoresence Microscopy--
106 Ba/F3
transfectants were harvested and washed with PBS at 4 °C. Cells were
then stained with a 1:100 dilution of rabbit anti-PRLr antiserum
developed by our laboratory and characterized elsewhere (40) for 1 h at 4 °C. After washing three times with PBS, bovine serum albumin,
0.1% sodium azide, the cells were incubated for 30 min with a 1:2000
dilution of fluorescein 5-isothiocyanate-conjugated goat anti-rabbit
secondary antibody. Cells were washed three times with PBS/bovine serum
albumin/sodium azide, fixed with PBS, 4% paraformaldehyde for 15 min
and resuspended in PBS/bovine serum albumin/sodium azide. Cellular
immunofluoresence was examined using a Zeiss Axioskop2
immunofluorescence microscope (Carl Zeiss, Inc., Thornwood, NY) and an
Apogee CCD camera (Axiom Research, Tucson, AZ). This antiserum
reactivity was specific for PRLr expression, since the addition of
competitive immunizing peptide was previously shown to inhibit PRLr
staining (40).
Scatchard Analysis--
Ligand binding affinities were
determined as described previously (41). Briefly, 106
transfected Ba/F3 cells were incubated with increasing concentrations of human [125I]PRL in a total volume of 100 µl of RPMI,
0.1% sodium azide. Nonspecific binding was estimated by incubating
separate tubes with unlabeled competitor PRL at a concentration 100 times greater than that of labeled ligand. Cells were incubated at
4 °C for 2 h with agitation and pelleted through an oil
gradient (90% dibutyl phthalate, 10% olive oil). Pellets were cut
from the tubes, and cell-associated [125I]PRL was
measured on a 1272 Clinigamma Quantitation of Ligand-induced Proliferation and Survival of PRLr
Isoform Transfectants--
To assess PRL-induced cellular
proliferation, 5 × 104 long and intermediate isoform
Ba/F3 transfectants were aliquoted in medium consisting of RPMI 1640 medium supplemented with sodium selenide, linoleic acid, insulin, and
transferrin (ITS+; Calbiochem, Bedford, MA) in the presence of 0-1
µg/ml human PRL or murine IL-3. After overnight culture, cells were
pulsed with 0.5 µCi of [3H]thymidine at 37 °C for
4 h. Incorporation of radiolabel was determined by
scintillography. To assess the viability of PRLr isoform transfectants,
Ba/F3 cells transfected with the PRLr intermediate isoform, the long
isoform construct, or control vector were plated at 106
cells/well in 2 ml of RPMI 1640 ITS+ with or without 10 ng/ml PRL.
Cells were harvested over a 72-h period, and the numbers of both dead
and viable cells were determined by trypan blue dye exclusion. The
percentage of viability was calculated by the number of live
cells/(live + dead cells) × 100%.
Immunoprecipitation and in Vitro Kinase Assays--
After PRL
stimulation (250 ng/ml), 2 × 105 CHO cells
transfected with PRLr isoforms in conjunction with Jak2 or Fyn
cDNAs (all expressed in vector pEF1-V5/HisA) were lysed and
immunoprecipitated overnight as described previously (11) using 3 µl
anti-Fyn (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or anti-Jak2
(Upstate Biotechnology, Lake Placid, NY) antibodies. Antigen-antibody
complexes were isolated by the addition of 50 µl of protein A beads.
After three washes with lysis buffer, immunoprecipitates were washed once with low salt buffer (10 mM Tris (pH 7.0), 100 mM NaCl, and 100 µM
Na3VO4). The protein A-Sepharose beads were
then suspended in 30 µl of autokinase buffer (25 mM Tris
(pH 7.0), 10 mM MnCl2, and 10 µCi of
[ Isolation of the Human Intermediate PRLr--
Although three
isoforms of the PRLr have been discovered in the rat (42), only one has
been found expressed in human tissues. Investigations from our
laboratory (39) and others (30) found evidence suggesting the existence
of at least one additional human isoform. To identify and characterize
these putative PRLr isoforms, reverse transcription PCR was performed
on cDNA generated from the breast cancer cell line T47D using
oligonucleotides homologous to the 5'- and 3'-ends of the human long
PRLr. Gel electrophoresis of the amplified cDNA revealed DNA
fragments of 1.3, 1.5, and 1.8 kilobases (Fig.
1, lane 4). Both
negative control reactions failed to amplify any DNA fragments,
suggesting that the bands identified were indeed generated from
cDNA template and not the result of chromosomal DNA contamination
(Fig. 1, lanes 1 and 2). The three
fragments were excised and cloned, and their DNA sequences were
determined. The 1.8-kilobase band was found to be the long PRLr isoform
previously described (38). The 1.5-kilobase fragment was an mRNA
splice variant coding for a truncated extracellular domain designated
In Vivo Expression of the Intermediate PRLr Isoform--
To
investigate the physiological activities of the intermediate isoform in
PRLr signaling, a eukaryotic expression vector containing the
intermediate PRLr cDNA was transfected transiently into CHO cells
and stably into the murine pro-B cell line Ba/F3. To serve as controls,
empty vector (negative control) and the long PRLr isoform (positive
control) were also used to transfect cells. Using a DNA probe
homologous to the extracellular domain common to both isoforms,
Northern analysis of the transfectants showed mRNA transcripts of
the predicted molecular mass for both long and intermediate
isoform-expressing clones (data not shown). The intermediate isoform
was also analyzed for its ability to be efficiently translated. The
cDNAs for both forms were subcloned into the vector pEF1V5/HisA,
which enabled the addition of a V5 epitope tag to the carboxyl-terminal
ends of both PRLr variants. Immunoblotting of lysates from CHO cells
transiently transfected with the constructs revealed proteins of the
correct molecular mass previously reported for the long form (85 kDa)
(43) and approximately 50 kDa for the intermediate isoform (Fig.
3). The predicted molecular mass for the
epitope-tagged intermediate PRLr is 32.7 kDa. Glycosylation of the
extracellular domain in a fashion similar to that found on the long
PRLr could account for the difference in the predicted and observed
mobilities, since treatment of both transfectants with tunicamycin
resulted in a decrease in apparent molecular mass (data not shown).
To confirm the expression of the intermediate PRLr on the cell surface,
stable Ba/F3 transfectants were stained with anti-PRLr antiserum and
examined for surface staining of the receptor (Fig. 4). Both long and intermediate PRLr
transfectants showed high levels of a speckled, cell surface staining
pattern compared with vector alone (Fig. 4, columns
1 and 2 versus column
3), while staining with preimmune serum only resulted in
background labeling of the transfectants (Fig. 4, row
2).
The Intermediate PRLr Isoform Binds Ligand with an Affinity
Comparable with the Long PRLr Isoform--
The rat intermediate PRLr
has been shown to bind ligand with a higher affinity than the rat long
form (37). To compare the ligand binding affinities of the human long
and intermediate variants, Scatchard analysis was performed on Ba/F3
transfectants. After adjusting for nonspecific binding of radiolabeled
PRL, similar dissociation constants and levels of surface expression
were observed for both isoforms; the long PRLr had a dissociation
constant (Kd) of 1.79 ± 0.22 nM,
while the intermediate isoform had a Kd of 1.64 ± 0.23 nM. Both transfectants also showed comparable
levels of surface expression (long = 6026 ± 517 receptors/cell versus intermediate = 6300 ± 674 receptors/cell). Unlike the differences in ligand affinity previously
observed for the comparable rat isoforms, both human isoforms showed
equivalent ligand binding.
The PRLr Intermediate Isoform Fails to Elicit Significant
Ligand-induced Proliferation--
Ba/F3 cells have been shown to
proliferate in response to PRL when transfected with rat long and
intermediate PRLr isoforms (44, 45). To examine the effects of human
intermediate PRLr homodimerization, transfectants were incubated with
increasing concentrations of human PRL and analyzed for DNA replication
by tritiated thymidine incorporation (Fig.
5A). As expected, Ba/F3 cells
expressing the long PRLr isoform proliferated in response to ligand,
while cells transfected with vector alone did not proliferate at any
PRL concentration. The intermediate PRLr could induce modest proliferation, but only at the highest concentration of PRL used (1 µg/ml). This lack of significant proliferation observed was not the
result of the transfection or selection process, since all three
transfectants stimulated with IL-3 showed comparable levels of
proliferation (Fig. 5B). Additionally, these results could
not be due to variability in cell surface expression or ligand binding
efficiency between the transfectants, since both the long and
intermediate isoforms showed comparable levels of surface expression
and similar Kd values. Taken together, the human
intermediate isoform is deficient in its ability to stimulate Ba/F3
cell proliferation in the presence of PRL.
The PRLr Intermediate Isoform Enhances Cell Viability--
While
the intermediate PRLr isoform was unable to induce proliferation of
Ba/F3 transfectants, this receptor variant may have an alternative
physiological role. For example, the long PRLr also mediates cell
survival and resistance to dexamethasone-mediated apoptosis (46, 47).
In addition, we have previously demonstrated that in the absence of
appropriate ligand, Ba/F3 cells die an apoptotic death that corresponds
to the measurement of trypan blue viability (46). To determine if the
human intermediate isoform may enhance cell survival, Ba/F3
transfectants were incubated with or without PRL over a 3-day period,
and cell viability was determined by trypan blue dye exclusion. Upon
the addition of ligand, cells transfected with either isoform showed
enhanced levels of cell viability as compared with vector-transfected
cells (Fig. 5C), while both long and intermediate
transfectants showed high levels of cell death in the absence of ligand
(Fig. 5D). In summary, although the intermediate isoform is
unable to invoke the complete signaling pathways necessary for cell
division, the intermediate isoform inhibits apoptosis in Ba/F3
transfectants, enhancing the survival of cells in the presence of ligand.
Protein-tyrosine Kinase Activation Differs between the Long and
Intermediate PRLr Isoforms--
Since the Ba/F3 intermediate isoform
transfectant was unable to induce proliferation upon ligand binding but
did enhance cell survival, we wished to examine if there were
differences in the proximal signaling pathways activated by the long
and intermediate prolactin receptors. The protein-tyrosine kinases
p59fyn and Jak2 are known to be activated upon ligand
stimulation of the PRLr (11, 12). To investigate whether the PRLr
intermediate isoform was capable of activating these proximal signaling
molecules, in vitro kinase assays were carried out using CHO
cells transiently transfected with constructs expressing these
molecules in conjunction with the PRLr isoform constructs. The
intermediate PRLr was capable of activating Jak2 in a temporal fashion
identical to the long PRLr (Fig.
6A), with maximal expression
occurring 15 min after the addition of ligand. In contrast to these
results, the long PRLr isoform exhibited high levels of Fyn activation,
while the ability of the intermediate isoform to stimulate Fyn was
greatly diminished (Fig. 6B). In summary, the pattern of
proximal protein-tyrosine kinase activation differed between the long
and intermediate isoforms, suggesting differences in proximal cell
signaling in response to ligand.
The Human PRLr Intermediate Isoform Shows Variable Tissue
Expression--
Previous studies have shown that the number of
isoforms and levels of PRLr expression vary between tissues in several
different species (28, 30, 31, 48). To determine if the intermediate isoform shows the same variability in expression between tissues, a dot
blot containing mRNA isolated from a variety of human tissues were
probed with cDNA fragments specific for either the long or intermediate isoforms (Fig. 7). To avoid
cross-hybridization, probes were generated that would hybridize only to
the mRNA transcripts encoding either the long or intermediate PRLr
isoforms. Relative levels of expression of both isoforms were compared
with those of the pituitary. Tissue expression of long PRLr mRNA
varied greatly between tissues, an observation previously reported in
the rat (48). The highest levels of expression of both isoforms were observed in the placenta, demonstrating over 6-fold more long PRLr and
5-fold more intermediate PRLr mRNA in comparison with the
pituitary. Aside from the high placental levels, mRNA expression between isoforms differed among the remaining tissues. For example, the
next three greatest amounts of long PRLr mRNA were found in the
adrenal gland, pituitary, and hippocampus. In contrast, the highest
levels of intermediate PRLr mRNA were found in the adrenal gland,
small intestine, and kidney. In summary, the intermediate form of the
PRLr shows significant variability in its expression between tissues,
and this pattern differs from that of the long form, suggesting a
differing physiological role for the new isoform.
While three PRLr isoforms have been identified and characterized
in the rat, only the long PRLr has been characterized in humans.
However, previous studies have strongly suggested the existence of
several human PRLr isoforms. For the first time, we report the
characterization of a novel, full-length human PRLr isoform isolated
from the human breast carcinoma cell line T47D. The human intermediate
isoform derives its name from the similarity it shares with the rat
intermediate PRLr. Unlike its rat counterpart, the human intermediate
PRLr results from an RNA processing event occurring at a consensus
splice junction. This isoform was termed the intermediate PRLr because
of the similarities it shares with the rat intermediate PRLr in both
the proximity of the splice junctions and sizes of the nucleotide
deletions observed (Fig. 8). Unlike the
rat intermediate PRLr, the splice also induces a frameshift after
residue 312, causing the addition of 13 heterologous amino acids and a
premature stop codon.
Immunoblotting of transiently transfected CHO cells expressing the
intermediate PRLr revealed a molecular mass of approximately 50 kDa,
compared with the 85-90-kDa-long PRLr observed by us and others (43).
Given that the extracellular domains of both human isoforms are
identical, it can be inferred the glycosylation patterns of both forms
may be similar. Therefore, the deletion of 573 base pairs resulting in
the loss of 30 kDa from the intracellular domain of the intermediate
PRLr would account for its apparent molecular mass. Efficient cell
surface targeting of the PRLr requires N-glycosylation (49).
Since asparagine residues believed to be glycosylated on the long PRLr
are conserved in the intermediate PRLr, we realized the intermediate
isoform would be expressed on the cell surface. As anticipated, Ba/F3
transfectants showed equivalent levels of long and intermediate PRLr on
their respective cell surfaces, and the intermediate PRLr bound ligand
with an affinity comparable with the long PRLr. This contrasts with the
rat long and intermediate isoforms in which the rat intermediate PRLr
bound ligand with a 3-fold higher affinity than the rat long PRLr
(37).
Ba/F3 cells were previously shown to be a reliable background to
measure cell proliferation, since the expression of the PRLr renders
them PRL-responsive (45, 50). The intermediate PRLr transfectants
exhibited no proliferation at physiological concentrations of PRL and
only modest [3H]thymidine incorporation at pharmacologic
concentrations of PRL. Ba/F3 cells transfected with the long PRLr, in
comparison, demonstrated robust proliferaton to physiologic
concentrations of PRL. In contrast to these results, the enhancement of
cell survival over a 72-h period was comparable between the
intermediate and the long PRLr. Taken together, these results suggested
that fundamental signaling differences existed between the two human
PRLr isoforms. In conjunction with the above proliferation studies,
transient transfections were undertaken to delineate differences in
proximal PRLr signaling between the two human PRLr isoforms. The
addition of PRL to intermediate PRLr-expressing transfectants resulted
in the activation of Jak2, while the activation of Fyn was severely
diminished. This was in contrast to the human long PRLr, which induced
the activation of both Jak2 and Fyn upon ligand binding. These
differences in proximal signaling may help explain the physiological
characteristics attributable to cells expressing the intermediate PRLr.
Previous mutational analysis of the PRLr by others (16, 18, 49) and us
(50) indicated that residues in the C terminus may be necessary for
maximal signaling and proliferation due to their associations with
Stat5 and Fyn. Mutation of C-terminal tyrosines in the rat intermediate
PRLr isoform affected Stat5-associated and Stat5-mediated gene
transcription (49). This is significant in that the human intermediate
PRLr is truncated by a frameshift upstream of these tyrosine residues,
resulting in an intracellular domain that lacks putative Stat5 binding motifs.
The lack of C-terminal tyrosines may also explain the poor activation
of Fyn via the intermediate PRLr and explain the altered mitogenic
response induced by the intermediate isoform. Fyn has been shown to
play an essential role in the platelet-derived growth factor receptor
(51), Ig (52), and T-cell receptor (53) signaling. In addition, we have
shown that Fyn is activated during PRL stimulation of Nb2 cells (11,
50). We previously reported that the truncation of the rat intermediate
PRLr at residue 322 or the mutation of the C-terminal
Tyr382 completely abrogated the mitogenic response, and
these two forms were incapable of activating Fyn (54). Taken together,
the lack of the C-terminal domain in the intermediate PRLr may preclude Fyn and Stat5 activation, thereby inhibiting the mitogenic response.
While it has been suggested that the proto-oncogene Cbl may play a role
in PRL-mediated Nb2 cell survival (57), several lines of evidence also
suggest that the activation of Jak2 via the intermediate PRLr may be
responsible for the enhanced survival of Ba/F3 transfectants. For
instance, IL-3-dependent 32Dcl3 cells transfected with a
dominant negative form of Jak2 exhibited decreased levels of Erk-2
kinase and accelerated apoptosis (58). A link between Jak2 inactivation
and apoptosis was also shown in human blood eosinophils (59). When
cells were treated with the Jak2 inhibitor tyrphostin B42 in the
presence of granulocyte-macrophage colony-stimulating factor, receptor
dimerization was unable to prevent eosinophil apoptosis. Last,
activated Jak2 has been implicated in increasing the expression levels
of Bcl-2 (60), a member of a family of genes thought to serve as
central regulators of programmed cell death (61, 62). A link between
PRLr signaling and the induction of another antiapoptotic gene, the
Bcl-2-associated protein Bag-1, has also been shown by our laboratory
(46). Ba/F3 transfectants overexpressing Bag-1 were rendered
IL-3-independent, while ligand stimulation and survival of Nb2 cells
was associated with increased Bag-1 levels. It is quite possible that
intermediate PRLr signaling, through Jak2, may increase Bcl-2 and/or
Bag-1 levels, explaining the enhanced survival of Ba/F3 transfectants. Future experiments will determine if signaling through the intermediate isoform can enhance Bag-1 expression.
Examination of the mRNA levels of intermediate PRLr as compared
with long PRLr revealed variable levels of PRLr isoform expression within different human tissues. Of the tissues examined, the highest levels of both long and intermediate PRLr mRNA were observed in the
placenta. Maaskant et al. (63) previously reported PRLr gene
expression in placental trophoblast and Western analysis detected six
molecular species, two of which were approximately the size of the
intermediate PRLr described herein. While the physiological function of
lactogenic binding proteins in the placenta is unknown, prolactin
receptors in uteroplacental tissues are believed to mediate effects of
lactogenic hormones on decidual function at midgestation (64, 65).
Given that both PRLr isoforms were found at high levels on these
tissues, regulation of hormone action during pregnancy in the placental
unit could result from PRLr isoform hetero- and/or homodimerization.
Of the three tissues examined from the gastrointestinal tract (stomach,
small intestine, and colon), the small intestine exhibited the greatest
expression of intermediate PRLr. This correlated with previously
described high levels of rat short PRLr expression (30). It is well
established that PRL plays an important role in the immune system
including an increase in the cytotoxic activities of natural killer
cells and the proliferation of lymphocytes from the spleen or lymph
nodes (66). It is possible that the intermediate PRLr may play a role
in the immunomodulation of the gut, since the intestinal tract is known
to be the first barrier against bacterial pathogens. Another function
of PRL in the small intestine is to regulate water and electrolyte
transport across the epithelium. The lack of PRL-induced responsiveness
in the colon (67) may be secondary to no intermediate PRLr mRNA and
very little long PRLr mRNA being detected in this segment of the
bowel. Taken together, the intermediate PRLr may play a role in both
the immunomodulation and osmotic balance of the gastrointestinal tract.
The adrenal gland exhibited high levels of both long and intermediate
PRLr message. Previous studies utilizing immunostaining and reverse
transcription PCR detected PRLr in all three zones within the adrenal
cortex and little labeling of the adrenal medulla (68). This suggests a
direct effect of PRL on adrenal cells, and in fact it was shown that
PRL increases steroidogenesis, enhancing the secretion of aldosterone
and cortisol in human adrenal cell cultures (68). One or both isoforms
may form part of a feedback mechanism in the adrenal gland, as cortisol
is known to inhibit PRL secretion.
Another organ that expressed high levels of intermediate PRLr
transcript was the kidney. Prolactin is known to induce ornithine decarboxylase activity in the kidney (69), and the low amount of
detectable long PRLr transcript observed suggests that the intermediate
PRLr may be capable of inducing this enzyme. On the other hand, the
kidney regulates systemic water electrolyte balance and is known to
play a major role in osmoregulation in amphibians and fish (70, 71).
There is sizable evidence supporting the existence of PRL binding sites
in the kidney (72-75), and PRLr mRNA was found in renal tubules
(76, 77). Indeed, both PRLr and PRL can be localized on the parietal
epithelium of Bowman's capsule (78). Taken together, this suggests an
autocrine/paracrine loop within the parietal epithelium, regulated by
one or both human PRLr isoforms, which may contribute to osmoregulation.
Previous studies of PRLr isoform expression have shown they are not
equally represented in all tissues and may in fact regulate the effects
of PRL through hetero- and homodimerization. In the mouse, for example,
examination of the transcripts of both short and long isoforms showed
variation of expression in a tissue-specific manner, depending on the
stage of the estrous cycle, pregnancy, and lactation (48, 79). This
variable expression suggests differences in the physiological roles of
PRLr isoforms and co-transfection experiments utilizing different PRLr
isoforms further support this hypothesis. Transient transfection of
BMGE cells with rat long and short forms showed an inhibition of
PRL-induced We thank Drs. Roy Duhe and Paul Stein for
providing Jak2 and Fyn constructs, respectively.
*
This study was supported in part by the National Institutes
of Health grants 2R01CA69294 (to C. V. C.) and 1F32DK09727 (to J. B. K.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF166329.
2
J. B. Kline and C. V. Clevenger,
manuscript in preparation.
The abbreviations used are:
PRL, prolactin;
PRLr, prolactin receptor;
PCR, polymerase chain reaction;
IL, interleukin;
CHO, Chinese hamster ovary;
PBS, phosphate-buffered
saline.
Functional Characterization of the Intermediate Isoform of the
Human Prolactin Receptor*
ABSTRACT
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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INTRODUCTION
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-casein gene products, respectively. PRLr dimerization
also induces the GRB2/SOS/Ras/Raf/MEK/MAPK signaling cascade,
ultimately activating several transcription factors necessary for cell
cycle progression including Myc, Jun, and T cell factor (19-21).
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-32P]dCTP in the presence of random
hexamers using the Oligo Labeling Kit (Amersham Pharmacia Biotech). The
probe was hybridized to the membrane at a final concentration of 100 ng/ml at 68 °C for 1 h in Express-hyb solution
(CLONTECH, Palo Alto, CA) as per the manufacturer's instructions. The blot was then washed three times in 2× SSC, 0.05% SDS at room temperature followed by two 20 min washes in 0.1× SSC, 0.1% SDS at 50 °C, followed by autoradiography.
-counter (EG & G Wallac, Akron, OH).
-32P]ATP). After 20 minutes at 30 °C, the
reactions were stopped by the addition of 2× Laemmli buffer with
mercaptoethanol, and the reaction products were analyzed by 10%
SDS-polyacrylamide gel electrophoresis followed by autoradiography.
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S1.2 In contrast, the
1.3-kilobase sequence revealed an mRNA splice variant coding for an
isoform with a deletion in the intracellular domain (Fig.
2A). This DNA sequence is most
likely the result of an RNA splicing event, since a consensus splice
site was present at the juncture between base pairs 1009 and 1582 (39).
The open reading frame is homologous to the long isoform up to base
pair 1009, where a deletion of 573 nucleotides occurs, juxtaposing base
pair 1009 to 1583 (Fig. 2B). While this isoform demonstrated 100% homology to the long form downstream of base pair 1582, the deletion caused a shift in the reading frame, altering the carboxyl amino acid sequence and generating a stop codon 13 residues after the
splice junction (Fig. 2B). Based on the proximity of the
splice junction to the gene deletion in the rat Nb2 intermediate
isoform (Fig. 8) (37), this PRLr variant was designated the human
intermediate PRLr.
View larger version (39K):
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Fig. 1.
Reverse transcription PCR of the human PRLr
intermediate isoform. PCR-amplified cDNA generated from T47D
mRNA using conserved 5' and 3' PRLr primers was loaded on a 1%
agarose gel, electrophoresed, and visualized by ethidium bromide
staining under UV light. Lane 1, T47D mRNA
without reverse transcriptase; lane 2, no
mRNA template; lane 3, chloramphenicol
acetyltransferase (CAT) mRNA (Life Technologies)
amplified with chloramphenicol acetyltransferase-specific primers;
lane 4, T47D mRNA plus reverse transcriptase.
L, the long PRLr isoform; S1, an alternative
splice variant deleting exons 4 and 5 of the extracellular domain;
I, the intermediate PRLr isoform.
View larger version (39K):
[in a new window]
Fig. 2.
Complete coding sequence of the human
intermediate PRLr transcript. A, open reading frame of
the intermediate isoform. The shaded area
indicates amino acid sequence divergence from the long PRLr isoform,
the boxed area indicates the transmembrane
region, and the underlined regions indicate the Box 1 and Box 2 motifs.
B, frameshift region of the intermediate PRLr isoform.
Numbers indicate nucleotide locations of the long form. The
arrow indicates the beginning of a 527-base pair deletion
and frameshift within the intermediate form.
View larger version (51K):
[in a new window]
Fig. 3.
Western analysis of PRLr transfectants.
2 × 105 CHO cells transiently transfected for 48 h with vector pEF1V5/HisA or PRLr isoforms were lysed, electrophoresed
on an 8% SDS-polyacrylamide gel electrophoresis gel, and transferred
to nitrocellulose. Isoforms were visualized by staining with a 1:1000
dilution of horseradish peroxidase-conjugated anti-V5 monoclonal
antibody (Invitrogen). Lane 1, pEF1V5/HisA CHO
transfectant; lane 2, long isoform CHO
transfectant; lane 3, intermediate isoform CHO
transfectant.
View larger version (13K):
[in a new window]
Fig. 4.
Surface expression of PRLr isoforms on Ba/F3
transfectants. 106 Ba/F3 transfectants were stained
with a 1:100 dilution of an anti-PRLr antiserum (upper
row) or preimmune serum (lower row)
followed by a 1:40 dilution of fluorescein 5-isothiocyanate-conjugated
goat anti-rabbit secondary antibody. Cells were fixed with 4%
paraformaldehyde/PBS and analyzed by immunofluorescent microscopy.
Left column, long form transfectant;
middle column, intermediate form transfectant;
right column, vector transfectant. Magnification: × 200.
View larger version (25K):
[in a new window]
Fig. 5.
The intermediate isoform enhances the
viability but not the proliferation of Ba/F3 transfectants. 5 × 104 Ba/F3 transfectants were incubated with 0-1 µg/ml
PRL (A) or IL-3 (B) for 24 h. Cells were
pulsed 4 h with 0.5 µCi [3H]thymidine, harvested,
and quantitated on a liquid scintillation counter. Wells containing
106 Ba/F3 transfectants were incubated with 10 ng/ml PRL
(C) or medium (D) in 2 ml of RPMI ITS+ over a
72-h period. Cells were harvested, and the percentage of viability was
determined by trypan blue dye exclusion.
View larger version (43K):
[in a new window]
Fig. 6.
Jak2 but not p59fyn is activated by
the PRLr intermediate isoform. CHO cells expressing PRLr receptor
isoforms in conjunction with Jak2 (A) or p59fyn
(B) were stimulated with 250 ng/ml human PRL for the
indicated times. Jak2 and Fyn were immunoprecipitated with
corresponding antisera and incubated with [ -32P]ATP
without exogenous substrates. After washing, the precipitates were
separated by 10% SDS-polyacrylamide gel electrophoresis and visualized
by autoradiography. The same amounts of the samples used in the
in vitro kinase assays were immunoprecipitated and
immunoblotted to show equal protein expression between samples
(lower panels).
View larger version (27K):
[in a new window]
Fig. 7.
Differential tissue expression of long and
intermediate isoform mRNA transcripts. cDNA probes
specific for the long (black bars) and
intermediate forms (white bars) were hybridized
to a whole tissue mRNA dot blot. Autoradiographs of the blot
were scanned, and the intensity of the signals was quantitated using
ImageQuaNT densitometry software (Molecular Dynamics). Signals were set
relative to expression observed in the pituitary gland.
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View larger version (22K):
[in a new window]
Fig. 8.
Comparison of the human intermediate PRLr
isoform with known human (A) and rat
(B) isoforms. ECD, extracellular
domain; 1, Box 1 motif; 2, Box 2 motif;
V, variable box motif. Numbers designate
positions of tyrosine residues. Black shading
indicates a shift in reading frame, causing divergence from the primary
amino acid sequence of the long isoform.
-casein gene transcription directly proportional to the
ratio of short to long form expressed (80). In other studies,
granulocyte colony-stimulating factor receptor/PRLr chimeras were used
to hetero- and homodimerize the intracellular domains of rat PRLr
isoforms (45). While Ba/F3 cotranfectants expressing only the rat
intermediate or long homodimers proliferated, heterodimers of
short/intermediate, short/long, and intermediate/long isoforms were
inactive. Thus, the observed variability in the expression of the human
long and intermediate PRLr isoforms reported here may represent a
physiologic mechanism through which the tissue-specific actions of this
receptor complex are regulated.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of Pathology & Laboratory Medicine, University of Pennsylvania Medical Center, 509 Stellar-Chance Labs, 422 Curie Blvd., Philadelphia, PA 19104. E-mail:
clevengc@mail.med.upenn.edu.
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