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J Biol Chem, Vol. 274, Issue 26, 18686-18692, June 25, 1999
From the Department of Cell Biology, Hagedorn Research Institute,
Niels Steensensvej 6, DK-2820 Gentofte, Denmark
Expression of the prolactin receptor (PRLR) gene
is increased in pancreatic islets during pregnancy and in
vitro in insulin-producing cells by growth hormone (GH) and
prolactin (PRL). The 5'-region of the rat PRLR gene contains at least
three alternative first exons that are expressed tissue-specifically
because of differential promoter usage. We show by reverse
transcription-polymerase chain reaction analysis that both exon 1A- and
exon 1C-containing PRLR transcripts are expressed in rat islets and
that human (h)GH, ovine (o)PRL, and bovine (b)GH increase exon 1A
expression 6.5 ± 0.8-fold, 6.8 ± 0.7-fold, and 3.9 ± 0.7-fold and exon 1C expression 4.8 ± 0.4-fold, 4.4 ± 0.6-fold, and 2.5 ± 0.7-fold, respectively. Expression of exon 1B
was not detectable. The transcriptional activities of reporter
constructs containing the 1A, 1B, or 1C promoter were found to be
22.8-fold, 2.7-fold, and 8.0-fold, respectively, above that of a
promoterless reporter construct when transfected into the
insulin-producing INS-1 cells. The transcriptional activity of the 1A
promoter construct was increased 8.9 ± 1.9-fold by 0.5 µg/ml
hGH. Responsiveness to hGH of the 1A promoter was localized to the
region from Prolactin (PRL)1 is a
pituitary hormone with pleiotropic effects influencing lactation,
reproduction, water-salt balance, behavior, immune regulation, growth,
and metabolism (1). One of the target tissues of PRL is the endocrine
pancreas where PRL and the related hormones growth hormone (GH) and
placental lactogen have been found to be potent growth factors for the
pancreatic The PRLR belongs to the same family as the GH receptor, and they are
part of the cytokine receptor superfamily characterized by their
ability to activate Janus kinases and STATs (signal transducers and
activators of transcription) (1). STATs are latent transcription factors that upon phosphorylation by Janus kinases dimerize and translocate to the nucleus, where they activate transcription via
binding of specific DNA elements termed The functional diversity of PRL is reflected by the wide distribution
of its receptor. Multiple isoforms of the PRLR have been identified
which vary in their sequence and in the length of their intracellular
domain (1). Furthermore, PRLR mRNAs have been found to display
heterogeneity in their 5'-untranslated region. Genomic analysis of the
5'-region of the rat and mouse PRLR genes has shown a complex
organization with the presence of at least five different first exons
that are alternatively spliced onto a common, noncoding exon 2 (17-19). The expression pattern of three of these first exons,
designated 1A, 1B, and 1C (18) or E12, E11, and
E13, respectively (17), has been studied in the rat and
found to be tissue-specifically restricted. Thus expression of exon 1A
is predominant in the liver, whereas exon 1B expression has been found
exclusively in the gonads. Exon 1C is widely expressed and strongly
up-regulated in mammary gland during lactation (17, 18). A major
transcription start site has been found for exon 1B in rat ovary, and
multiple transcription start sites have been identified for both exon
1A and exon 1C in rat liver (17). Transcription of these first exons is
initiated by separate promoters, which are utilized in a
tissue-specific manner. The 1A promoter has been found to be active in
hepatoma cells and inactive in cells derived from mammary gland. In the proximal part of this promoter we have identified a binding site for
the liver-enriched transcription factor hepatocyte nuclear factor 4 (HNF4), which may be involved in transcriptional activation of the 1A
promoter in liver (18). In the 1B promoter from the rat, a binding site
for steroidogenic factor-1 has been identified and shown to be
essential for activation of this promoter in gonadal cells (20).
However, the mouse 1B promoter lacks a functional steroidogenic
factor-1 element and is inactive in gonadal cells. This indicates that
the 1C promoter is important for PRLR gene transcription in many
tissues across species. Recently, binding sites for the widely
expressed transcription factors CAAT-box/enhancer-binding protein The aim of the present study was to determine PRLR promoter usage in
pancreatic islets and to elucidate the molecular mechanism by which GH
and PRL regulate PRLR gene expression in insulin-producing cells.
Hormones and Cell Culture--
Recombinant hGH (3 IU/mg) was
obtained from Novo Nordisk (Gentofte, Denmark). Bovine (b)GH (0.81 IU/mg) and ovine (o)PRL (30 IU/mg) were purchased from UCB (Brussels,
Belgium). Pancreatic islets were isolated from 3-5-day-old Wistar rats
(Møllegård, Lille Skensved, Denmark) by the collagenase method (3).
The islets were cultured free-floating in bacteriological plastic Petri
dishes (Nunc, Roskilde, Denmark) for 3-5 days in RPMI 1640 medium
supplemented with 10% newborn calf serum, 20 mM Hepes, 100 units/ml penicillin, and 100 µg/ml streptomycin. Before the addition
of hormones, the islets (5,000 islets/dish) were cultured for 7 days in
15 ml of RPMI 1640 supplemented with 0.5% normal human serum. INS-1
cells (22) were cultured in complete medium (RPMI 1640 medium
supplemented with 10% heat-inactivated fetal calf serum, 50 µM 2-mercaptoethanol, 100 units/ml penicillin, and 100 µg/ml streptomycin) at 37 °C in a humidified atmosphere containing
5% CO2 in air. Once a week the cells were detached by
trypsinization, and the next passage was seeded 1:4.
RNA Extraction and cDNA Synthesis--
Islets were
stimulated for 24 h in the absence or presence of 500 ng/ml hGH,
bGH, or oPRL and then harvested by centrifugation. The islets were
lysed in RNAzol (Cinna/Biotecx Laboratories International Inc.,
Houston, TX), and total RNA was extracted using the single step
acid-guanidium-thiocyanate-phenol-chloroform method (23) as described
by the manufacturer. RNA concentrations were determined from optical
absorbence at A260 nm. According to
the manufacturer's protocol, cDNA was synthesized from 1 µg of
islet RNA in a volume of 25 µl using the RNase H Primers--
The total PRLR mRNA pool in the rat islets was
determined by a primer set with exon 10-derived sequences: 5'-GCT GAT
GTG TGC AAG CTA GCC GGA A (forward); 5'-GAC ACC TTG GCA TAC TCC TTA CTG G (reverse). To detect PRLR mRNA species containing exon 1A, 1B, or
1C sequences, the following three primers were used as forward primers:
1A, 5'-AGC GAG CTG GAT TCT AGG GAA ACA T; 1B, 5'-AGA GCC ATT CTC CAG
TAC TGT GAA T; 1C, 5'-TAA AAT CCC CAG ACG CCG GGT CTT C. The sequence
of the common reverse primer was derived from exon 3: 5'-TTG TGG ATC
TCA GGT TTC CCT GGT G. The expected lengths of the various PCR products
were as follows: PRLR, 340 bp; 1A, 329 bp; 1B, 495 bp; 1C, 396 bp.
Furthermore, a primer set with sequences derived from the rat
glucose-6-phosphate-dehydrogenase (G6PDH) cDNA was included in the
PCRs as an internal control: 5'-GAC CTG CAG AGC TCC AAT CAA C
(forward); 5'-CAC GAC CCT CAG TAC CAA AGG G (reverse) (24). The
expected size of this PCR product was 214 bp.
RT-PCR Analysis--
PCR was carried out in 50-µl reactions
using 3 µl of cDNA as template. The PCRs contained 50 mM KCl; 10 mM Tris-HCl, pH 9.0; 1.5 mM MgCl2; 40 µM dATP, dGTP, and
dTTPs; 20 µM dCTP; 2.5 µCi of 3,000 Ci/mmol
[ Plasmids--
The three luciferase reporter constructs pGL2-1A,
pGL2-1B, and pGL2-1C were generated as follows. The 5'-flanking regions of PRLR exons 1A ( Transient Transfection and Dual Luciferase Reporter (DLR)
Assay--
INS-1 cells were seeded in 24-well plates (300,000 cells/well) in complete medium. The following day the cells were
transiently transfected by the use of LipofectAMINE (Life Technologies,
Inc.) essentially as recommended by the manufacturer. Briefly, 1.5 µl of LipofectAMINE and 0.5 µg of DNA (250 ng of one of the firefly luciferase reporter plasmids, 10 ng of pRL-SV40 plasmid (internal control), and 240 ng of pUC18 plasmid (carrier)) were used per well.
The cells were transfected overnight in Opti-MEM (250 µl/well). The
medium was changed to fresh RPMI 1640 medium containing 0.5% fetal
calf serum and incubated for 24 h in the presence or absence of
hormones as indicated. The cells were harvested in 100 µl of passive
lysis buffer (supplied with the DLR assay system, purchased from
Promega), and the cell extracts were stored at Nuclear Extracts--
INS-1 cells (approximately 2 × 107) were cultured in 150-mm tissue culture dishes for 2 days in complete medium. The medium was changed to fresh medium
containing 0.5% fetal calf serum, and after 16 h the cells were
incubated for 15 min in the absence or presence of 500 ng/ml hGH, bGH,
or oPRL as indicated. The cells were washed in phosphate-buffered
saline and lysed in hypotonic buffer A (20 mM Hepes, pH
7.9; 10 mM KCl, 1 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 0.5 mM AEBSF, 1 mM Na3VO4,
1 µg/µl leupeptin, 1 µg/µl aprotinin, 20% glycerol) containing
1% Triton X-100. The nuclei were scraped off and collected by
centrifugation after which they were extracted for 30 min on a rocking
bench in hypertonic buffer B (buffer A with 400 mM NaCl).
The extracts were centrifuged at 20,000 × g for 30 min, and aliquots of the supernatant were frozen in liquid nitrogen and
stored at Electrophoretic Mobility Shift Analysis (EMSA)--
The
double-stranded oligonucleotide 1A-GLE (see Fig. 4A) was
radiolabeled in a fill-in reaction using [ Exons 1A and 1C, but Not 1B of the Rat PRLR Gene Are Expressed in
Insulin-producing Cells in a GH- and PRL-regulated Way--
We have
employed quantitative RT-PCR analysis to determine the expression
levels of exons 1A, 1B, and 1C in insulin-producing cells. We first
used a primer set specific for exon 10 of the PRLR gene. Exon 10 is
present in transcripts encoding the long form of the receptor; and
because the long form of the PRLR is the predominant form expressed in
islets (11), the majority of PRLR mRNA species in these cells are
recognized by this primer set. Using cDNA prepared from cultured
rat islets, the expected 340-bp PCR product was detected easily (Fig.
1A, lane 1), and the level was found to be up-regulated by stimulation with bGH, oPRL,
or hGH (Fig. 1A, lanes 2, 3, and
4, respectively). It should be noticed that in rodents bGH
and oPRL activate GH and PRL receptors, respectively, whereas hGH is
known to activate both types of receptors. Correlated to the internal
control G6PDH, the level of PRLR mRNA was increased 3.3 ± 0.9-fold by bGH, 5.8 ± 0.1-fold by oPRL, and 6.1 ± 1.2-fold
by hGH (Fig. 1B). These results are in accordance with those
obtained by RNase protection assay using a probe specific for the long
form of the PRLR (11). Using three upstream primers specific for exons
1A, 1B and 1C and a common downstream primer specific for exon 3 (see
Fig. 2A) we detected exons 1A-
and 1C-specific PCR products with the expected sizes of 329 and 396 bp,
respectively (Fig. 2B, lanes 1-4 and
9-12, respectively), whereas exon 1B expression was not
detected (Fig. 2B, lanes 5-8). Exon 1A
expression was found to be up-regulated 3.9 ± 0.8-fold by bGH,
6.8 ± 0.7-fold by oPRL, and 6.5 ± 0.7-fold by hGH, whereas
exon 1C expression was found to be up-regulated 2.5 ± 0.4-fold by
bGH, 4.4 ± 0.6-fold by oPRL, and 4.8 ± 0.7-fold by hGH
(Fig. 2C). The expression and hormonal regulation of exons
1A, 1B, and 1C were found to be similar in INS-1 cells (data not
shown), suggesting that results obtained in the latter cells regarding
the mechanism of PRLR gene regulation will apply to primary Transcriptional Activity and Hormonal Regulation of the 1A, 1B, and
1C Promoters in INS-1 Cells--
To determine the transcriptional
activity of the 1A, 1B, and 1C promoters in insulin-producing cells, we
transiently transfected INS-1 cells with firefly luciferase reporter
genes under the transcriptional control of the 5'-flanking regions of
exons 1A, 1B, and 1C, respectively. As shown in Fig.
3A, the pGL2-1A construct
containing the region of the 1A promoter from Localization of GH/PRL-responsive Element(s) in the 1A
Promoter--
To identify the region(s) of the 1A promoter which
contained putative GH/PRL-responsive element(s) we analyzed a series of 5'-deletion mutants of the pGL2-1A construct which, as mentioned above,
contained the region of the 1A promoter from Identification of a STAT5-binding Element within the
GH/PRL-responsive Region of the 1A Promoter--
Within the
GH/PRL-responsive region of the 1A promoter, we identified a putative
GLE with the sequence 5'-TTCTAGGAA located at position
To identify the proteins present in the hGH-induced complex, nuclear
extract from hGH-stimulated INS-1 cells was preincubated with
STAT5a-specific antibody, STAT5b-specific antibody, an antibody recognizing both STAT5a and STAT5b, or a control antibody. As shown in
Fig. 4C, the complex was partly supershifted by the
anti-STAT5a antibody (lane 2) and almost completely
supershifted by the anti-STAT5b (lane 3) and anti-STAT5a+b
antibodies (lane 4), whereas the control antibody
(lane 5) left the complex unchanged. These results indicate that hGH induces binding of STAT5a and STAT5b to 1A-GLE. In contrast to
the anti-STAT5 antibodies, antibodies directed against STAT1 or STAT3
did not recognize the hGH-induced complex binding to 1A-GLE (data not
shown). Activation of PRL and GH receptors individually via stimulation
with either oPRL or bGH, respectively, induced bandshift patterns
identical to that induced by hGH. Furthermore, the intensities of the
complexes induced by hGH, bGH, and oPRL were similar (Fig.
4C, lanes 1, 6, and 11,
respectively). Also, the supershift pattern obtained by the addition of
anti-STAT5a, anti-STAT5b, and anti-STAT5a+b antibodies to nuclear
extracts from either bGH- or oPRL-stimulated cells were similar to that observed with nuclear extracts from hGH-stimulated cells (Fig. 4C, lanes 7-9 and 12-14,
respectively, compared with lanes 2-4).
Mutation of the STAT5-binding Element Renders the 1A Promoter
Construct Unresponsive to GH/PRL--
To assess the role of 1A-GLE in
the responsiveness of the 1A promoter to GH and PRL, we employed
site-directed mutagenesis on the pGL2-1A construct. We changed the
1A-GLE sequence from TTCTAGGAA to TTCTAGGCT because EMSA
had shown that mutation of these 2 bases rendered the 1A-GLE unable to
bind STAT5 (see Fig. 4B). As shown in Fig.
5, the basal promoter activity of the
pGL2-1A-mutGLE construct was found to be similar to that of the pGL2-1A
construct, when expressed as luciferase ratios (0.09 ± 0.02 and
0.06 ± 0.03, respectively). In contrast, the promoter activity of
only the wild type construct and not that of the mutated construct was found to be increased by hGH (0.53 ± 0.18 and 0.14 ± 0.05, respectively).
The present study shows that PRLR transcripts containing either
exon 1A or exon 1C but not 1B are present in insulin-producing cells.
Furthermore, PRL and GH increase the steady-state levels of both
transcripts. In transfected INS-1 cells, the 1A and 1C promoters but
not the 1B promoter were transcriptionally active. The increase in exon
1A expression induced by GH and PRL could be explained by induction of
the 1A promoter. Furthermore, mutation of the STAT5-binding element
located in the proximal part of the 1A promoter was found to abolish
responsiveness to GH and PRL, suggesting that these hormones activate
the 1A promoter via the STAT5 signaling pathway. STAT5 has been found
to mediate GH- and PRL-induced expression of the rat insulin 1 gene
(16), and thus STAT5 activation seems to be important for the
regulation of gene expression by PRL and GH in insulin-producing cells.
It is possible that STAT5 activation is also involved in the
stimulatory effects of PRL and GH on It has been suggested that at least some of the phenotypic differences
of mice, in which either the STAT5a or the STAT5b gene has been
disrupted, could be ascribed to differences in the expression levels of
STAT5a and STAT5b rather than isotype-specific functions (15). Our gel
shift data indicate that STAT5b is more abundant than STAT5a in the
GH/PRL-induced complex binding 1A-GLE. Whether this reflects a higher
level of STAT5b expression relative to STAT5a in the INS-1 cells
remains to be determined. The more potent induction of exon 1A
expression observed with PRL compared with GH during long term
stimulation might be because PRLR expression is increased by PRL,
whereas GHR expression is unaffected by GH (11).
The transcriptional activity of the pGL2-1C promoter construct was
found to be unaffected by GH and PRL stimulation when transfected into
INS-1 cells. This construct contains the region of the 1C promoter from
GH- and PRL-induced up-regulation of exons 1A and 1C expression might
not be restricted to the pancreatic islets. In the liver where both are
expressed, GH and PRL have been reported to increase PRLR gene
expression (26-29). Furthermore, PRL is thought to increase the PRLR
mRNA level in the mammary gland during lactation where we have
observed a marked increase in the level of exon 1C expression (18).
Interestingly, we found that the transcriptional activity of the 1A
promoter reporter construct pGL2-1A was not increased by either GH or
PRL when transiently transfected into Chinese hamster ovary cells
expressing GH or PRL receptors, respectively (data not shown). Because
transfection of the 1A promoter into these cells produced significant
luciferase activity (18), these data might indicate that responsiveness
of this promoter to GH and PRL is tissue-specifically regulated.
The absence of exon 1B expression in insulin-producing cells confirms
the previously observed gonad-restricted expression of this first exon
of the PRLR gene (17, 18). In contrast, exon 1C expression has been
found in all tissues examined, namely gonads, liver, and mammary gland
(17, 18). The present finding that exon 1C is also expressed in
insulin-producing cells supports a common utilization of the 1C
promoter in a wide variety of tissues. The transcriptional activity of
the pGL2-1A promoter construct was found to be 3-fold higher than that
of the pGL2-1C promoter construct in INS-1 cells. High levels of exon
1A expression have been found previously only in the liver, and we have
identified a binding site in the 1A promoter for the liver-enriched
transcription factor HNF4 which may mediate hepatic exon 1A expression
(18). HNF4 is expressed also in pancreatic In summary, PRLR mRNA transcripts containing either exon 1A or exon
1C as first exon are expressed in rat pancreatic islets. GH and PRL
were found to increase the expression of both of these PRLR mRNA
species in these cells. Although the molecular mechanism of the
hormonal regulation of exon 1C expression is still unclear, GH and PRL
up-regulate exon 1A expression via activation of STAT5a and STAT5b
which stimulates the transcriptional activity of the 1A promoter by
binding to the GLE identified in the proximal part of the promoter. It
is likely that up-regulation of PRLR expression in the We thank Dagny Jensen and Jannie Rosendahl
Christensen for excellent technical assistance and Nils Billestrup and
Ying Chiu Lee for helpful discussion.
*
The Hagedorn Research Institute is an independent basic
research component within the Health Care Discovery Division of Novo Nordisk A/S.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 abbreviations used are:
PRL, prolactin;
GH, growth hormone;
PRLR, PRL receptor;
STAT, signal transducer and
activator of transcription;
GLE,
Regulation of Prolactin Receptor (PRLR) Gene Expression in
Insulin-producing Cells
PROLACTIN AND GROWTH HORMONE ACTIVATE ONE OF THE RAT PRLR GENE
PROMOTERS VIA STAT5a AND STAT5b*
,
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225 to +81 with respect to the transcription start site.
This region contains the sequence TTCTAGGAA that by gel retardation
experiments was shown to bind the transcription factors STAT5a and
STAT5b in response to stimulation by hGH, oPRL, or bGH. Mutation of
this 
-activated sequence-like element completely abolished
transcriptional induction of the 1A promoter by hGH. Our results
suggest that GH and PRL increase the levels of exon 1A- and
1C-containing PRLR mRNA species and furthermore that the transcriptional activity of the 1A promoter is increased via activation of STAT5a and STAT5b.
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-cell and to enhance insulin production and glucose
sensitivity of these cells (2-6). The stimulatory effects of PRL, GH,
and placental lactogen are thought to play an important role in the
adaptive growth and function of the -cells during conditions of
increased insulin demands. Insulin-producing cells express both GH and
PRL receptors (7-10), and in particular the long form of the PRL
receptor (PRLR) is up-regulated during pregnancy with a sustained
elevation observed during lactation (11). PRL and GH have been shown to stimulate PRLR mRNA expression in insulin-producing cells in
vitro (7, 11, 12). The up-regulation of PRLR expression may be an
important mechanism in the adaptation of the -cells to the increased
insulin demand during pregnancy. Failure in this pathway may lead to
gestational diabetes.
-activated sequence-like elements (GLEs) having the consensus sequence TTCNNNGAA (13). GH and
PRL have been shown to activate a subset of STAT proteins, namely
STAT1, STAT3, STAT5a, and STAT5b (1, 14). Although the physiological
significance of GH- and PRL-induced STAT1 and STAT3 activation is still
unclear, the generation of STAT5a and STAT5b knockout mice showed that
the two STAT5 isoforms have essential roles in a wide range of
biological actions of PRL and GH (15). In vitro STAT5a and
STAT5b have been found to mediate GH- and PRL-induced activation of a
number of tissue-specifically expressed genes, including the rat
insulin 1 gene (16).
and Sp1 have been reported to regulate the activity of the 1C promoter
in hepatic and gonadal cells (21). It is likely that the differential
usage of the PRLR promoters enables the complex tissue-specific
regulation of PRLR gene expression.
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reverse transcriptase Superscript II and random primers (Life Technologies, Inc.). The cDNA samples were stored at 20 °C
after the addition of 50 µl of 0.1% Triton X-100.
-33P]dCTP (Amersham Pharmacia Biotech); 10 pmol of
each primer; and 2.5 units of Ampli Taq Gold polymerase
(Perkin-Elmer/Roche). A single denaturing step at 94 °C/10 min was
followed by 25 cycles as given: 94 °C/30 s; 55 °C/1 min;
72 °C/1.5 min. PCR products were separated on 6% denaturing
polyacrylamide gels containing 7 M urea, 130 mM
Tris, 80 mM boric acid, and 0.25 mM EDTA. The gels were dried and visualized by autoradiography. In addition the gels
were exposed to PhosphorImage storage screens that were scanned by
Molecular Dynamics PhosphoImager series 400 (Molecular Dynamics,
Sunnyvale, CA), and band intensities were calculated using the program
Image-Quant (Molecular Dynamics).
999/+81), 1B (311/+339), and 1C (417/+200), when expressed in relation to transcription start sites of the individual exons, were inserted into the plasmid pGL2-Basic (Promega, Madison, WI) containing the coding region of the firefly luciferase gene, as described previously by Møldrup et al. (18).
Furthermore, the 1A promoter 5'-deletion constructs (462, 404,
356, 255, 225, and 83) were generated by exonuclease III
digestion of the pGL2-1A construct (18). The QuikChangeTM
site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to
introduce mutations in the GLE of the pGL2-1A construct. The sequences
of the two complementary, mutagenic primers were as follows with the
mutated bases underlined: 5'-CAA AAT GAG TTC TAG TCA TAA
AGA ATA TCA G; 5'-CTG ATA TTC TTT ATG ACT AGA ACT CAT TTT
G. The resulting construct (pGL2-1A-mutGLE) was sequenced to ensure
that the two point mutations were introduced correctly. The plasmid
pRL-SV40 containing the coding region of the Renilla luciferase gene under the transcriptional control of the SV40 early enhancer/promoter was purchased from Promega.
80 °C. Firefly and
Renilla luciferase activities of the cell extracts were
measured with a Turner Designs model 20/20 luminometer (Promega) using the DLR assay system. According to routine, 30 µl of cell extract was
measured for a 15-s period at a sensitivity level of 80%. The levels
of firefly luciferase activities were in the range of 0.23-1.23 units
(pGL2-Basic), 4.41-11.73 units (pGL2-1A), 0.79-1.46 units (pGL2-1B),
and 3.57-3.73 units (pGL2-1C); the levels obtained from
mock-transfected cells were in the range of 0.09-1.37 units. The
levels of Renilla luciferase activities obtained from
pRL-SV40-transfected cells were in the range of 83-418 units with the
majority of the samples having activities in the range of 100-200.
Renilla luciferase activities obtained from mock-transfected
cells were in the range of 20-31 units. When calculating the
luciferase ratio (firefly luciferase activity above Renilla
luciferase activity) the corresponding mock activities were first
subtracted. Each DLR assay was repeated three or four times with
duplicate samples.
80 °C. Protein concentrations were measured using
Bio-Rad protein assay.
-32P]dATP
(Amersham Pharmacia Biotech) and DNA polymerase (Klenow fragment) and
used as probe. Nuclear extracts (10 µg) were incubated for 30 min at
30 °C with 20 fmol of probe in a 20-µl reaction containing 20 mM Hepes, pH 7.9; 50 mM NaCl; 1 mM
MgCl2; 1 mM EDTA; 1 mM
dithiothreitol; 10% glycerol; and 0.1 µg/µl
poly(dI-dC)·poly(dI-dC). Free and bound probe were separated on a 5%
polyacrylamide gels containing 2% glycerol and 0.25 × TBE (25 mM Tris-HCl, 25 mM boric acid, and 0.25 mM EDTA; pH 7.9). The gels were dried and exposed to
autoradiography for 1-2 days. In competition studies, 200 or 2,000 fmol corresponding to 10- and 100-fold molar excess, respectively, of
unlabeled 1A-GLE was added to the binding reaction. Alternatively, unlabeled double-stranded mut1A-GLE oligonucleotide (see Fig. 4A) was used as competitor. In supershift studies, nuclear
extracts were preincubated at 4 °C for 1 h with 1 µl of
either one of the following antibodies: anti-STAT5a and anti-STAT5b
(Zymed Laboratories Inc. Laboratories, San Francisco,
CA); anti-STAT5a+b, which recognizes both STAT5a and STAT5b; and
anti-c-Myc, which was included as control antibody (Santa Cruz
Biotechnology, Santa Cruz, CA).
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-cells
as well.
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Fig. 1.
RT-PCR analysis of the hormonal regulation of
PRLR mRNA expression in cultured rat islets. Panel
A, islets isolated from newborn rats were cultured for
24 h in the absence (control, lane 1) or presence of
500 ng/ml bGH (lane 2), oPRL (lane 3), or hGH
(lane 4). cDNA was synthesized from total RNA, and
RT-PCR was performed with a primer set generating a PCR product of 340 bp specific for exon 10 of the PRLR gene. A primer set generating a PCR
product of 214 bp specific for G6PDH was included as an internal
control. Shown is a representative autoradiograph of the PCR products
separated by denaturing polyacrylamide gel electrophoresis with the
molecular weight marker shown in lane 5. Panel
B, quantification of the effects of bGH, oPRL, and hGH on
the PRLR mRNA level. The levels of the 340-bp PRLR-specific PCR
product were normalized to G6PDH levels, and the results are expressed
as fold induction (mean ± S.D., n = 3) compared
with control levels.
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Fig. 2.
RT-PCR analysis of the expression and
hormonal regulation of PRLR exons 1A, 1B, and 1C in cultured rat
islets. Panel A, schematic representation of
the genomic structure of the 5'-end of the rat PRLR gene with exons
indicated by filled boxes. The locations of the three
upstream primers specific for exons 1A, 1B, and 1C and the common
downstream primer specific for exon 3 are indicated by
arrows. Panel B, RT-PCR was performed
and visualized as described in Fig. 1 using cDNA from unstimulated,
control islets (lanes 1, 5, and 9) or
from islets stimulated with 500 ng/ml bGH (lanes 2,
6, and 10), oPRL (lanes 3,
7, and 11), or hGH (lanes 4,
8, and 12). Primer sets specific for exon 1A
(lanes 1-4), 1B (lanes 5-8), and 1C
(lanes 9-12) were included in the PCRs together with a
primer set specific for the internal control G6PDH. The molecular
weight marker is shown in lane 13. Panel
C, quantification of the effects of bGH, oPRL, and hGH on
the levels of exon 1A- (left panel) and 1C- (right
panel) containing transcripts. The levels of exons 1A- and
1C-specific PCR products (329 and 396 bp, respectively) were normalized
to G6PDH levels, and the results are expressed as fold induction
(mean ± S.D., n = 4) compared with the
corresponding control levels.
999 to +81 relative to
transcription start site (18) showed relatively strong promoter
activity when analyzed by DLR assay. The transcriptional activity of
the pGL2-1A construct was 22.8-fold higher than that of the
promoterless pGL2-Basic construct into which the various PRLR promoter
fragments have been cloned (luciferase ratios: 7.6 × 102 ± 2.0 × 102 and 0.3 × 102 ± 0.3 × 102, respectively). In
contrast the promoter activity of the pGL2-1B construct containing the
region from 311 to +339 of the 1B promoter was only 2.7-fold higher
than that of pGL2-Basic (0.8 × 102 ± 0.2 × 102). The pGL2-1C construct containing the region from
417 to +200 of the 1C promoter had intermediate promoter activity
that was 8.0-fold higher that of pGL2-Basic (2.4 × 102 ± 0.9 × 102). The
transcriptional activity of the pGL2-1A construct was induced 9.2-fold
by hGH (70 × 102 ± 28 × 102).
Both oPRL and bGH stimulated the transcriptional activity of the
pGL2-1A construct; and although the level of induction obtained with
oPRL was similar to that seen with hGH, bGH was found to be less potent
(data not shown). Furthermore, the effect of hGH was
dose-dependent with a 2.6 ± 0.7-fold induction
obtained with as little as 1 ng/ml hGH (Fig. 3B). In
contrast, hGH had no significant effect on the transcriptional activity
of the pGL2-Basic, pGL2-1B, and pGL2-1C constructs (0.3 × 102 ± 0.1 × 102, 1.3 × 102 ± 0.5 × 102, and 3.6 × 102 ± 1.7 × 102, respectively) as
shown in Fig. 3A.
View larger version (18K):
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Fig. 3.
Transcriptional activity and GH/PRL
responsiveness of the 1A, 1B, and 1C promoters of the rat PRLR gene in
insulin-producing cells. INS-1 cells were transiently
cotransfected with the indicated firefly luciferase reporter constructs
and the internal control construct pRL-SV40, which contained the
Renilla luciferase gene. After culture in the presence or
absence of the indicated amounts of hGH, cell lysates were prepared and
DLR assays performed as described under "Experimental Procedures."
Panel A, firefly luciferase reporter constructs
containing either no promoter (pGL2-Basic) or the 1A ( 999/+81;
pGL2-1A), 1B (311/+339; pGL2-1B), and 1C (417/+200; pGL2-1C)
promoters were transfected into INS-1 cells that were then cultured for
24 h in the absence () or presence (+) of hGH (500 ng/ml). The
results (mean ± S.D., n = 3-4) are expressed as
luciferase ratios (see "Experimental Procedures"). Panel
B, INS-1 cells were transfected with the pGL2-1A firefly
luciferase reporter construct and cultured for 24 h in the absence
(0 ng/ml) or presence of 1, 5, 10, 100, or 500 ng/ml hGH as indicated.
The results (mean ± S.D., n = 4) are luciferase
ratios expressed as fold induction by hGH compared with that obtained
with transfected cells cultured in the absence of hGH. Panel
C, the following 5'-deletion constructs of the pGL2-1A
plasmid, which contained the region of the 1A promoter from 999 to
+81, were transfected into INS-1 cells: 462, 404, 356, 255,
225, and 83. The cells were cultured for 24 h in the absence
() or presence (+) of hGH (500 ng/ml). The results (mean ± S.D., n = 4) are luciferase ratios expressed as a
percent of that obtained with cells transfected with the pGL2-1A
construct and cultured in the absence of hGH.
999 to +81. As shown in
Fig. 3C, the 462, 404, and 356 deletion constructs were similar to the full-length pGL2-1A construct with respect to both
basal transcriptional activity and hormonal responsiveness. When
expressed as percent of pGL2-1A, the activities were 110 ± 17%,
149 ± 32%, and 148 ± 17% in the absence and 978 ± 192%, 747 ± 53%, and 833 ± 79% in the presence of hGH.
Although the basal transcriptional activities of the 255 and 225
constructs were reduced to 49 ± 15% and 14 ± 3%,
respectively, these two constructs still responded to hGH with
increased promoter activity (358 ± 142% and 62 ± 20%,
respectively). In contrast, the transcriptional activity of the 83
construct was not significantly higher than that of the promoterless
pGL2-Basic construct neither in the absence nor presence of hGH (5 ± 1% and 6 ± 2%, respectively). These results suggest that a
GH/PRL-responsive element is present in the region from 225 to
+81.
156 to 148
(Fig. 4A). This putative GLE
is homologous to STAT5 binding sites known to mediate GH and/or PRL
responsiveness to other genes such as the serine protease inhibitor 2.1 gene and the insulin gene. To determine whether the putative GLE of the
1A promoter is able to bind nuclear proteins in a GH- and PRL-inducible
way, gel retardation experiments were performed using the
double-stranded oligonucleotide termed 1A-GLE as probe (Fig.
4A). With nuclear extract from hGH-stimulated INS-1 cells, a
prominent band was observed, which was not present in nuclear extract
from unstimulated cells (Fig. 4B, lanes 3 and
2, respectively). Formation of this hGH-induced complex was
partially inhibited by the addition of a 10-fold molar excess and
almost completely inhibited by the addition of a 100-fold molar excess
of unlabeled 1A-GLE oligonucleotide to the binding reaction (Fig.
4B, lanes 4 and 5, respectively). In
contrast, the addition of a 10- or 100-fold excess of the
oligonucleotide mut1A-GLE (see Fig. 4A) did not decrease the
intensity of the protein complex (Fig. 4B, lanes
6 and 7, respectively), showing that binding of the
complex to the 1A-GLE is highly specific.
View larger version (24K):
[in a new window]
Fig. 4.
GH- and PRL-induced binding of STAT5a and
STAT5b to the GLE of the 1A promoter. Panel
A, the sequence of the 1A promoter region from 162 to
142 is shown with the putative GLE in bold. Furthermore,
the sequences of the two oligonucleotides 1A-GLE and mut1A-GLE used in
EMSA as unlabeled competitors or radiolabeled probe (1A-GLE) are shown.
The 2 bases mutated in the mut1A-GLE oligonucleotide are
underlined. Panel B, the 1A-GLE probe
was incubated either in the absence (lane 1) or presence of
nuclear extracts (NE) prepared from INS-1 cells, which was
either unstimulated (lane 2) or stimulated with hGH for 15 min (lanes 3-7). Unlabeled 1A-GLE oligonucleotide was
included in the binding reaction as competitor (COMP) at
10-fold or 100-fold molar excess in lanes 4 and
5, respectively, whereas in lanes 6 and
7 unlabeled mut1A-GLE oligonucleotide was included at
10-fold and 100-fold molar excess, respectively. Panel
C, the 1A-GLE probe was incubated with nuclear extracts
isolated from INS-1 cells stimulated for 15 min with hGH (lanes
1-5), bGH (lanes 6-10), and oPRL (lanes
11-15). Before the addition of probe, nuclear extracts were
preincubated with antibodies (ab) recognizing either STAT5a
(STAT5a; lanes 2, 7, and 12),
STAT5b (STAT5b; lanes 3, 8, and
13), or STAT5a and STAT5b (STAT5a+b; lanes 4,
9, and 14). Alternatively, nuclear extracts were
preincubated with control antibody (lanes 5, 10,
and 15). EMSA was performed as described under
"Experimental Procedures," and the autoradiographs shown are
representative of two independent experiments. The arrow
indicates the migration of the 1A-GLE·STAT5 complex.
View larger version (27K):
[in a new window]
Fig. 5.
The GH/PRL-responsiveness of the 1A promoter
is abolished by mutation of the 1A-GLE. INS-1 cells were
transiently transfected with firefly luciferase reporter constructs
containing the 1A promoter fragment from 999 to +81 with the GLE
either left unaltered (pGL2-1A) or mutated (pGL2-1A-GLEmut).
Cotransfections were performed with the internal control plasmid
pRL-SV40. The cells were cultured for 24 h in the absence () or
presence (+) of hGH before harvest. DLR assays were performed, and the
results (mean ± S.D., n = 3) are expressed as
luciferase ratios.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cell proliferation and insulin
secretion because the phenotype of STAT5-deficient mice demonstrated an
essential role for these two proteins in a wide range of physiological
responses associated with GH and PRL (15). GH and PRL were equally
potent in activating STAT5 binding in INS-1 cells. These data are in agreement with the finding that in INS-1 cells GH induced nuclear translocation of STAT5 in a manner similar to that observed with PRL
(25). Although hGH is known to activate DNA binding of STAT1 and STAT3
in insulin-producing cells (16), these transcription factors were not
found to be present in the 1A-GLE-binding complex, indicating that they
are not mediating GH/PRL-induced activation of the 1A promoter. A
STAT5a-specific antibody partially supershifted the GH/PRL-induced
complex, whereas an almost complete supershift was obtained with a
STAT5b-specific antibody. Because STAT5 has yet to be shown to form
heterodimers with other members of the STAT family, our gel shift data
suggest that the GH/PRL-induced complex binding to the 1A-GLE consists
of STAT5b homodimers and STAT5a+b heterodimers.
417 to +200, which has been reported to mediate high levels of
transcriptional activity in cells of either gonadal or hepatic origin
(21). The lack of regulation by hGH might thus indicate that the
putative GH/PRL-responsive elements are not located within the region
from 417 to +200. Sequence analysis of the region from 1027 to + 351 did, however, not reveal any homology to known STAT5 binding sites,
which opens the possibility that the activity of the 1C promoter might
be induced by other GH- and PRL- regulated transcription factors.
Alternatively, GH and PRL might increase the level of exon
1C-containing PRLR transcripts by a mechanism other than
transcriptional activation of the 1C promoter.
-cells, but whether HNF4 is involved in expression of exon 1A in insulin-producing cells is
presently unknown. HNF4 seems to play an important role in the
-cells because subjects carrying mutations in the HNF4 gene have
been found to develop maturity onset of diabetes in the young resulting
from defects in -cell function (30).
-cells during
pregnancy is caused by increased levels of exon 1A- and/or
1C-containing PRLR transcripts, and thus failures in the pathways
regulating the expression of exons 1A and 1C may lead to gestational
diabetes from a lack of increased -cell mass and function, which
normally compensate for the increased insulin demand during pregnancy.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 45-44-439-081;
Fax: 45-44-438-000; E-mail: edg{at}hagedorn.dk.
ABBREVIATIONS
-activated sequence-like element;
HNF4, hepatocyte nuclear factor 4;
h, b, and o prefix, human, bovine,
and ovine, respectively;
PCR, polymerase chain reaction;
bp, base pair(s);
G6PDH, glucose-6-phosphate-dehydrogenase;
RT, reverse
transcription;
DLR, dual luciferase reporter;
AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride;
EMSA, electrophoretic
mobility shift analysis.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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