Inefficient Secretion of Human H27A-Prolactin, a Mutant That Does Not Bind Zn2+
Zhenyu Sun,
Min S. Lee,
Harrison K. Rhee,
Joanne M. Arrandale and
Priscilla S. Dannies
Department of Pharmacology, Yale University School of
Medicine, New Haven, Connecticut 06510
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ABSTRACT
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Human PRL binds Zn2+, but
the function of the binding is not known. We investigated the effect on
PRL production in pituitary cells by obtaining clones of
GH4C1 cells stably
transfected with human H27A-PRL, a mutant that does not bind
Zn2+. Unexpectedly, clones transfected with the
mutant human PRL made little rat PRL. Untransfected
GH4C1 cells made
between 0.5 to 10 µg rat PRL/105 cells in
24 h. Clones transfected with vector alone (four of four), wild
type human PRL (six of six), or with human K69A-PRL (two of two) made
amounts of rat PRL in the same range. Clones transfected with human
H27A-PRL (five of five) made 0.0030.1 µg rat
PRL/105 cells in 24 h, and the production
of rat PRL mRNA was reduced. Human H27A-PRL was not efficiently
secreted; 2040% newly synthesized H27A-PRL was degraded by 60 min,
and there was usually a delay in release of newly synthesized H27A-PRL.
Reduction of rat PRL production is not mediated through the PRL
receptor, because no sequences for the receptor in
GH4C1 cells were
detected by RT-PCR. Proteins involved in folding, such as BiP, were not
specifically elevated in the H27A-PRL clones. In transient
transfections, in which cells have not undergone selection, we found no
evidence for disulfide-bonded aggregates of the mutant protein. The
results indicate that Zn2+ binding stabilizes
PRL in the secretory pathway; the instablility of the mutant protein
may trigger effects that suppress rat PRL production directly or that
indirectly result in selection of clones with low rat PRL production.
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INTRODUCTION
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Zinc ions have been recognized as playing an important structural
role in many proteins (1), including the ability to stabilize insulin
in a hexamer, the form in which it is stored in secretory granules.
Binding Zn2+ is important for insulin storage because a
mutant of human insulin that does not bind Zn2+ well is not
properly processed (2). Zn2+ binding also may be important
for storage of other proteins concentrated in secretory granules. Human
GH binds Zn2+ (3). Binding of Zn2+ affects the
properties of human GH in solution; Zn2+ causes human GH to
form dimers and increases its stability, properties that have been
proposed to be important in storing GH in a concentrated form in
secretory granules (3). Whether it plays such a role in intact cells
has not been shown.
Human PRL, which has a structure like that of GH (4, 5), also binds
Zn2+ (6). Histidine 18 in human GH is required to bind
Zn2+ (3), and the topologically equivalent histidine 27 is
required in PRL. The two hormones differ, however, in the effects of
Zn2+ binding. PRL does not require Zn2+ binding
for lactogenic activity as human GH does, and the ability to bind
Zn2+ and to self-associate in solution are not coupled in
PRL as they are in human GH (6). Loss of histidine 18 in human GH
reduces Zn2+ binding and dimer formation (3). Loss of
histidine 27 in human PRL reduces Zn2+ binding, but not the
ability to self-associate in the presence of Zn2+ (6).
Although the binding site for Zn2+ that requires histidine
is not necessary for PRL to associate in a cell-free system, it may be
necessary for proper processing and packaging of PRL in cells, and we
have investigated this question by expressing human H27A-PRL in
GH4C1 cells, which are a rat pituitary cell
line.
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RESULTS
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In the same way in which we had previously isolated clones that
expressed human wild type PRL (7), we isolated stably transfected
clones of GH4C1 cells that expressed human
H27A-PRL. Unexpectedly, clones transfected with human H27A-PRL produced
reduced amounts of rat PRL (Table 1
). The
amounts of rat PRL made by untransfected GH4C1
cells varies in different experiments, and in ten experiments with
untransfected cells performed during the time the transfected clones
were analyzed, the amounts of rat PRL in the cultures ranged from
0.510 µg/105 cells in 24 h. All clones transfected
with vector alone, human wild type PRL, or human K69A-PRL, which were
obtained at the same time as the H27A-PRL clones, produced amounts
of rat PRL within the range of the untransfected cells. All clones
transfected with human H27A-PRL made less rat PRL, from 0.0030.1
µg/105 cells in 24 h. These values were obtained by
RIA; we also confirmed qualitatively by immunoblots that clones H27A6
and H27A8 contained much less rat PRL than
GH4C1 cells (not shown).
The decrease in rat PRL production was accompanied by a decrease in rat
PRL mRNA. Northern analysis of RNA extracted from
GH4C1 cells and from a clone, H27A6, indicated
there was less rat PRL mRNA in the transfected clone (Fig. 1
). The filter was assayed subsequently
with cyclophilin to demonstrate that we had recovered RNA from the
transfected clone. In Fig. 1
, the amount of cyclophilin mRNA was higher
in the transfected clone, but in a second experiment it was not. In
each case, rat PRL mRNA was reduced more than 30-fold in the
transfected clone.

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Figure 1. Northern Analysis of mRNA from Equal Numbers of
GH4C1 Cells and a Clone Transfected with Human
H27A-PRL
Top panel, rat PRL; bottom panel, the same
filter anaylzed for cyclophilin.
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GH4C1 cells in the absence of estradiol,
insulin, and epidermal growth factor store little rat PRL relative to
the amount they secrete, usually less than 5% of the amount secreted
in 24 h; when treated with hormones, cells store 15% or more (7).
We attempted measurements of rat PRL stored in clones transfected with
human H27A-PRL, but found no consistent results, even in separate
experiments using the same clone; intracellular PRL in control or
treated cells varied from 530% of the amount secreted in 24 h.
The variation was in contrast to our previous studies with clones
expressing human wild type PRL and other mutants, where the results
were consistent (7).
This type of analysis is a measure of storage if the protein is stable.
We examined the stability of H27A-PRL to determine whether
intracellular degradation might account for the variability we
encountered. In clone H27A6, we detected incorporation of
35S-labeled amino acids during a 10-min pulse only into
human PRL (Fig. 2
). In these series of
experiments, PRL was precipitated with concentrations of antisera to
rat PRL sufficient to precipitate both rat and human PRL completely,
and we determined that the band we detected was human PRL by its
mobility, and, in separate experiments, by using dilutions of antisera
specific to each species. The inability to detect rat PRL synthesis,
although it is detected in the medium, reflects the small amounts of
rat PRL that this clone usually made as well as the comparatively fewer
methionines in rat compared with human PRL.

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Figure 2. Fate of Newly Synthesized Human H27A-PRL
Cells were incubated for 10 min with 35S-labeled amino
acids, lysed at indicated intervals after the 10-min pulse, and PRL
immunoprecipitated, and the immunoprecipitate was subjected to
electrophoresis. Bottom panel, Radioactivity detected in
the position corresponding to human PRL in the cells (inside) and in
the medium (outside). Top panel, Quantification of
[35S]PRL. , [35S]PRL in the medium; ,
[35S]PRL in the cells.
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We did not detect newly synthesized human H27A-PRL in the medium for
times up to 90 min in the experiment shown in Fig. 2
. The lack of
detection of secreted H27A-PRL was not caused by degradation in the
medium because we incubated recombinant H27A-PRL with the cells in the
conditions that we use for the pulse-chase procedure, and we recovered
all the added hormone, measured by RIA. The lack of secretion was not
seen in all experiments; in five of six experiments with this clone and
two of three experiments with clone H27A8, we detected no secretion
during a 90-min chase period, but in one experiment with each clone,
there was detectable secretion at 30 min, as we found with rat PRL and
human wild type PRL (Fig. 3
).

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Figure 3. Fate of Newly Synthesized Human Wild Type and Rat
PRL
In an independent experiment, rat and human [35S]PRL were
also stable, but relatively more rat PRL, 40% of the total, was
secreted by 90 min. Bottom panel, Radioactivity detected
in the positions corresponding to human PRL and rat PRL. Middle
panel, Quantitation of human [35S]PRL; top
panel, quantification of rat [35S]PRL. ,
[35S]PRL in medium; , [35S]PRL in cells;
, total [35S]PRL.
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Intracellular human H27A-PRL was not completely stable. In the
experiment shown in Fig. 2
, there was 20% less
[35S]H27A-PRL at the end of the chase period than at the
end of the 10-min pulse. The decrease was reproducible; the amount of
[35S]H27A-PRL decreased 2040% by 60 min after the
pulse in all six experiments. The time that we first detected the
decrease was more variable, ranging from 2060 min after the pulse. We
did not detect such decreases in the clones expressing wild type human
PRL (Ref. 7 and Fig. 3
). The variation in amount and time of
degradation and secretion of H27A-PRL is likely to explain the
variability we encountered when we attempted to measure storage.
We determined whether human H27A-PRL was present in a regulated pathway
by stimulating release using high K+ concentrations to
depolarize the cell and stimulate Ca2+ entry (8).
Perifusing the cells with high K+ concentrations increased
human PRL release 2-fold (Fig. 4
). The
magnitude of the stimulation is similar to the 2- to 4-fold increases
that we detected in untransfected GH4C1 cells
at this time (7). The mutant therefore has the ability to enter a
regulated pathway.

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Figure 4. Stimulation of Human H27A-PRL Release by High
Concentrations of Potassium Chloride in Perifused Cells
Four minute fractions were collected, and the flow rate was 1 ml/8 min.
The delay time before added components appear in the medium is 10
min.
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The decrease in rat PRL production caused by expression of H27A-PRL
does not appear to be caused by simply interfering with folding or
stability of rat PRL, since rat PRL synthesis and mRNA production were
reduced, and amounts of rat PRL were decreased as measured by
[35]S-amino acid incorporation, RIA, and immunoblots.
The possible mechanisms for reducing rat PRL mRNA include a feedback
cycle activated inappropriately by H27A-PRL to reduce rat PRL
production or by unwitting selection for a property in addition to G418
resistance. The most obvious way for feedback to occur is through the
PRL receptor. We determined whether the PRL receptor was present in
GH4C1 cells by RT-PCR using primers that would
detect the long and the short form of the receptor. We were easily able
to detect appropriate PCR bands using 1 µg total RNA from Nb2 cells
but did not detect any in 1 µg RNA from GH4C1
cells in several experiments. Mixing equal amounts of RNA from
GH4C1 cells and Nb2 cells did not reduce the
ability to form product from Nb2 cells (Fig. 5
). We also mixed 106
GH4C1 cells with 105 Nb2 cells
before extracting the RNA and found no difference in the amount of
product compared with 105 Nb2 cells extracted alone (not
shown). The GH4C1 cells therefore do not
interfere with the extraction of RNA or the RT-PCR assay and do not
contain enough mRNA for either form of the receptor to be detectable.
It appears unlikely that feedback is occurring through the PRL
receptor.

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Figure 5. Products Formed by RT-PCR to Assay for the Presence
of the Short Form of the Rat PRL Receptor (Lanes 1 to 6) and the Long
Form (Lanes 7 to 11)
In the first lane, an Nb2 PRL receptor plasmid was used as a control.
Lanes 2 and 7, 1 µg Nb2 RNA; lanes 3 and 8, 1 µg untreated
GH4C1 RNA: lanes 4 and 9, 1 µg
hormone-treated GH4C1 RNA; lanes 5 and 10, 1
µg each Nb2 and untreated GH4C1 RNA; lanes 6
and 11, 1 µg each Nb2 and hormone-treated
GH4C1 RNA.
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A second mechanism by which feedback may occur is through communication
between the secretory pathway and the nucleus. Misfolded proteins in
the endoplasmic reticulum result in a signal that increases
transcription of proteins that assist in folding in the secretory
pathway, including GRP78 (glucose-regulated protein 78, also known as
BiP) (9, 10). We examined proteins known to reside in the endoplasmic
reticulum to determine whether they were induced specifically in clones
expressing human H27A-PRL. There was no increase in protein disulfide
isomerase (Fig. 6
and Table 2
). Three other proteins, GRP78, GRP94,
and calreticulin, may be somewhat increased compared with
GH4C1 cells, but were not reproducibly
different from cells transfected with human wild type PRL (Fig. 6
and
Table 2
). Production of H27A-PRL therefore does not appear to trigger
specifically the pathway that activates GRP78.

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Figure 6. Immunoblots of Several Proteins Located in the
Endoplasmic Reticulum in GH4C1 Cells, Two
Clones That Make Wild Type Human PRL (Wt-hPRL), and Two Clones That
Make Human H27A-PRL (H27A-hPRL)
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Selection for clones with properties in addition to resistance to G418
may occur if the transfected product interferes with cell growth in a
manner that can be bypassed. A simple means for expression of H27A-PRL
to cause selection may be that it initially folds so poorly in the
cells that it causes a disadvantage during selection. We have therefore
used transient transfections of GH4C1 cells to
determine how H27A-PRL behaves in unselected cells. We found transient
transfections have been useful only for qualitative conclusions because
of the low transfection efficiency and the variation among replicates.
The procedure that gave the best production of human PRL resulted in
expression of ß-galactosidase in 0.5 to 3% of the cells. Conditions
that gave higher transfection efficiencies resulted in substantially
reduced human PRL production. Variation in the amount of human PRL
produced in replicate cultures was as much as 2-fold, and
cotransfection with ß-galactosidase for normalization did not improve
this variation.
Although the information gained was only qualitative, these experiments
showed that there was no extensive misfolding of H27A-PRL. Both wild
type and the mutant were synthesized in a 10-min pulse, both were
present 90 min after the pulse, and small amounts of each form were
secreted (Fig. 7
). Expression of either
form has no effect on production of rat PRL, according to RIA
measurement, but the percentage of cells transfected is so small that
we would not predict that there would be an effect. We also performed
immunoblots on cell extracts run under reducing or nonreducing
conditions (Fig. 8
). We found no evidence
for disulfide-bonded aggregates; both human wild type and mutant PRL
ran as monomer in the presence or absence of reducing agents.

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Figure 7. Immunoprecipitates of PRL from Cells Transiently
Transfected with Human Wild Type or H27A-PRL After a 10-min Pulse with
35S-Labeled Amino Acids or an 80-min Chase
In this experiment, antibody to human PRL was used at a dilution that
precipitated all human PRL, but not all the rat PRL in the cells.
Because of the low transfection transficiency, most rat PRL is made in
cells that do not express human PRL. Two other experiments gave similar
results.
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Figure 8. Immunoblots of Cells Transiently Transfected with
Human Wild Type or H27A-PRL or a Mixture of the Two
The antibody to human PRL was used at a dilution that did not
cross-react with rat PRL standard at the amounts used (100 ng/gel), but
the amount of rat PRL in the cells was so much greater than human PRL
that it is detected, as seen in control cultures, or cultures treated
with Lipofectamine alone. The last three lanes are, in order, extracts
from cultures treated with 3 µg human wild type PRL DNA, 3 µg
H27A-PRL DNA, or 1.5 µg wild type plus 1.5 µg H27A-PRL DNA to keep
the total amount of DNA in the transfections constant. The gel in the
upper panel was run under reducing conditions, and the
one in the lower panel was run under nonreducing
condtions. The band at the top of the gel is a
nonspecific band detected in cell extracts.
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DISCUSSION
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The original purpose of this investigation was to determine
whether the ability to bind Zn2+ was necessary for
transport and secretion of PRL. Zn2+ has been shown to
reduce solubilization of PRL from dense cores of granules (11). Both
human GH and human PRL bind Zn2+, and Zn2+ has
been detected in granules containing PRL or GH (12). Zn2+
was not detected in all granules; whether the variation actually exists
or is a result of the staining technique has not yet been resolved.
Human GH is more stable in the presence of Zn2+ (3), and
Cunningham and co-workers therefore predicted Zn2+ binding
would enhance stability of the stored form of these hormones, which is
exactly what we found.
Human H27A-PRL, which has a greatly reduced ability to bind
Zn2+ (6), folds correctly, since it is transported through
the secretory pathway and is recognized in a RIA. It is also
transported in a regulated pathway where release is stimulated by
depolarization. The major effect of the H27A mutation appears to be on
the stability of PRL; in the pulse chase experiments, some of the newly
synthesized H27A-PRL was degraded, and there was usually a delay in the
secretion of newly synthesized hormone. The conditions of the pulse
chase experiments with medium changes and the resulting fluctuations in
factors, such as pH and temperature, may affect stability of the mutant
protein more markedly than wild type. The instability of the mutant
protein, induced by the experimental conditions, may interfere with
transport through the secretory pathway.
Although the instability of the H27A mutant in cells was predicted, we
did not expect the expression of endogenous rat PRL would be reduced in
the clones. Such a finding may be analogous to certain cases of GH
deficiency in which the presence of a mutant GH gene suppresses
secretion of the product of the normal gene (13, 14, 15). A mutation in
vasopressin that causes it to be dominant negative for secretion has
also been described (16). The mechanisms by which secretion of a normal
protein is reduced by a mutant protein are not known, although direct
interactions between normal and mutant proteins during folding so that
they are not correctly transported have been suggested. Results with
H27A-PRL demonstrate that, at least in this case, the cause is more
complex because the production of PRL mRNA is reduced as well. In
addition, when we examined the production of human wild type and
H27A-PRL in cells that had not undergone selection procedures, we found
no marked differences between the two; therefore, gross misfolding and
aggregation of H27A-PRL is unlikely to be a problem.
We found no evidence for the PRL receptor in
GH4C1 cells by PCR techniques, which indicates
there is not a possibility for feedback mediated through the receptor.
We had previously proposed that the PRL receptor might play a direct
role in causing PRL concentration into dense core granules, as an
explanation for the effects of mutations in human PRL on rat PRL
storage (7); our inability to detect the PRL receptor eliminates this
mechanism and raises the possibilities of indirect mechanisms, such as
selection or feedback, for the effects on storage as well.
Communication from the secretory pathway to the nucleus is known to
occur through several different mechanisms. Unfolded proteins in the
endoplasmic reticulum activate a transmembrane kinase that results in
increased transcription of chaperones, such as GRP78; this pathway has
been extensively investigated in yeast (10, 17, 18, 19, 20). Unfolded proteins
in the endoplasmic reticulum also activate an interferon-inducible,
double-stranded RNA-dependent protein kinase and the transcription
factor NF-
B, processes that appear to involve Ca2+
(21, 22, 23). The response to unfolded proteins is complex and involves
several pathways; although we did not find specific induction of GRP78,
other responses may have occurred. We found no evidence for massive
misfolding of H27A-PRL, such as disulfide-bonded aggregates, but the
instability that we detected may cause stress for the cells. Because
the mutant is not completely stable in cells, it may require more use
of chaperones and consumption of ATP to fold and maintain correct
conformations as it traverses the secretory pathway. Such increased
activity may result in a signal to the nucleus that directly regulates
synthesis of endogenous PRL or that indirectly results in selection of
clones that produce less rat PRL.
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MATERIALS AND METHODS
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Isolation of Stable Transfectants
The cDNA for human PRL was in pcDNA-NEO (7). Histidine 27 in
human PRL was mutated to alanine by PCR, using the primer extension
technique of Ho et al. (24), and for primers, SP6, T7,
5'-TCCGCCTACATCCATAACCTCTCC-3', and 5'-GTTATGGATGTAGGCGGACAGGAC-3'.
Lysine 69 was mutated in the same way, using SP6, T7,
5'-CCCGAAGACGCGGAGCAAGCC-3' and 5'-TTGGGCTTGCTCCGCGTG-3'. Synthesis of
primers and complete sequencing of all constructions to confirm they
were correct was done by the W. M. Keck Foundation Biotechnology
Resource Laboratory at Yale University.
All transfectants were isolated in the same way. Cells were transfected
by electroporation, and stable clones were obtained by growth in 0.5
mg/ml G418. Visible colonies appeared between 2 and 3 weeks. The medium
from colonies was screened for human PRL, and those that had
accumulated the most hormone were selected and expanded. In the
transfection with the empty vector, the colonies were randomly
selected. Assays for PRL production, expressed as amount per cell
number, were begun soon after the clones were expanded. Cells were
subcultured once a week, and assays for rat and human PRL were
performed within early passages for all clones.
For induction of PRL storage, cells were incubated in medium containing
15% gelding serum alone or plus 1 nM estradiol, 300
nM insulin, 5 nM epidermal growth factor, as
previously described (25).
Pulse Chase Procedure
Cells were treated with 1 nM estradiol, 300
nM insulin, and 5 nM epidermal growth factor
for 7 days. Labeling was performed in Hams F10 nutrient mixture with
0.1% horse serum, 1 mM NaHC03, and 20
mM HEPES, pH 7.5. After rinsing, cells were incubated with
400 µC Expre35S35S Protein Labeling Mix,
(Dupont New England Nuclear, Boston, MA) for 410 min and then in
medium with 1.5 mM methionine and cysteine for additional
time. Cells were lysed in 10 mM Tris-HCl, pH 7.5, 150
mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 1
mM EDTA plus 2.5 µg/ml leupeptin, 2.5 µg/ml pepstatin,
1 µg/ml aprotinin, 1 mM benzamidine, 0.4 mM
phenylmethylsulfonylfluoride (PMSF), 0.5% SDS, and 0.25% BSA. Medium
and lysate were incubated with antiserum for 18 h at 4 C. For most
experiments, we used rabbit anti-rat PRL from Arnel (New York, NY) at a
concentration (1:100) that was sufficient to precipitate both rat and
human PRL. Where indicated, we used anti-human PRL from NIH (PRL-IC-5)
at a concentration, 1:600, that precipitated all the human PRL in the
culture, but not all the rat PRL. At these concentrations, the antisera
recognize denatured PRL. The immunoprecipitates were collected with
Protein A-Sepharose beads (1.875 mg/sample; Pharmacia, Piscataway, NJ)
and washed three times with 50 mM Tris-HCl, pH 7.5, 150
mM NaCl, 0.1% NP40, 0.25% gelatin, 1 mM EDTA
and three times with 10 mM Tris-HCl, pH 7.5, and 0.1% NP40
for 30 min per wash. PRL was eluted from the beads by boiling for 5 min
in 50 mM Tris, pH 7.0, 4% glycerol, 2% SDS, and 10%
ß-mercaptoethanol, followed by 60 C for 15 min. PRLs were resolved by
SDS gel electrophoresis (7). Human PRL migrates more slowly than rat
PRL (7). Rat and human PRL produced by the clones were also
distinguished in preliminary experiments by the ability to be
precipitated by diluted concentrations of their respective antisera, as
well as the RF values (the distance migrated relative to
the front).
Immunoblots
Cells were treated as described above, the cells
collected, an aliquot removed for counting, and the rest centrifuged
and dissolved in loading buffer (100 mM Tris-HCl, pH 6.8,
4% SDS, 0.02% bromophenol blue, 20% glycerol, 10%
ß-mercaptoethanol, and 200 mM dithiothreitol), and heated
at 100 C for 5 min, followed by 60 C for 15 min. Samples from equal
numbers of cells were run on 12.5% polyacrylamide gels at 120 V.
Proteins were transferred from gels to Immobilon Membranes (Millipore,
Bedford, MA) at 300 mA for 1.5 h. Membranes were incubated with
antibody overnight, followed by incubation with rabbit antimouse
immunoglobulin when monoclonal antibodies were used, and finally with
10 µCi [125I]Protein A (specific activity, 8.5
µCi/µg, Dupont New England Nuclear) for 1 h. Antisera were
from Affinity Bioreagents (Neshanic Station, NJ) for proteins other
than PRL. Antisera for human PRL was that for immunocytochemistry from
NIH. A Molecular Imager system (Bio-Rad, Hercules, CA) was used to
detect and quantitate the bound radioactivity.
Perifusion
Cells cultured on Cytodex II beads (Pharmacia) were perifused as
previously described (25).
RIA
Rat and human PRL were detected by RIA. Reference preparations
NIDDK-pPRL-RP.1 and NIDDK-rPRL RP-3 were used for standards and were
provided by the National Hormone and Pituitary Program, the National
Institutes of Diabetes and Digestive and Kidney Diseases, the National
Institutes of Child Health and Human Development, and the US Department
of Agriculture. Rat and human [125I]PRL were from Dupont
NEN. Human H27A-PRL does not completely displace human
[125I]PRL in the assay (6); the samples were measured in
a range where displacement occurs, and the standard curves were
parallel. Each assay was specific for rat or human PRL and did not
recognize the other species.
Northern Analysis
Total RNA was prepared by an acid-guanidinium
thiocyanate-phenol-chloroform technique (26), subjected to
electrophoresis, and transferred to nitrocellulose. RNA was hybridized
to a rat PRL probe from Dr. Richard Maurer (University of Oregon),
labeled with 32P by random priming.
RT-PCR
Cells were disrupted in 6 ml 4 M guanidinium
isothiocyanate, 25 mM sodium citrate, pH 6.5, 0.5%
sarcosyl, 1.1 M ß-mercaptoethanol and passed three times
through a 25-gauge needle. The homogenate was then layered onto 4 ml
5.7 M CsCl, 25 mM sodium acetate, pH 6.0.
Solutions were centrifuged at 175,000 x g for 21
h at 22 C. The supernatants were aspirated and the RNA pellets were
washed once with 50% ethanol and then resuspended in H2O.
Total RNA was added to 1.25 µg oligo(dT)12-18primer
(GIBCO, Grand Island, NY), 0.5 mM deoxynucleotide
triphosphates, 10 U of rRNasin (Promega, Madison, WI), 500 U of
SuperScript II RNAse H- RT (GIBCO), 50 mM
Tris-HCl, pH 8.3, 75 mM KCl, 3 mM
MgCl2, 10 mM DTT in a total reaction volume of
50 µl and incubated at 42 C for 15 min. The RNA template was digested
with 2.7 U of ribonuclease H (GIBCO) at 37 C for 15 min. Five
microliters of reverse transcription reaction were added to a total
volume of 100 µl containing 20 mM Tris-HCl, pH 8.4, 50
mM KCL, 1.5 mM MgCl2, 0.5
µM of each primer, and 2.5 U of Taq DNA
polyerase (GIBCO). PCR was performed for 30 cycles with a denaturing
temperature of 94 C for 1 min, an annealing temperature of 65 C for 2
min, and an elongation temperature of 72 C for 2 min. The forward
primer for both the long and short form of the receptor was
5'-CATGGATACTGGACTAGATGGAGC-3'. The reverse primer for the long form
was 5'-CTCAGCAGCTATTCAGACTTG-3', to give a 250-bp fragment. The reverse
primer for the short form was 5'-TCCTATTTGAGTCTGCAGCTTCAGTAGTCA-3', to
give a 335-bp fragment. The plasmid containing sequences for the PRL
receptor used as a control was a gift from Lee Yuan Yu-Lee (27).
Transient Transfections
Hormone-treated cells were transfected with Lipofectamine
(GIBCO), using 2.5 x 105 cells, 3.2 µg DNA, 9.2
µl Lipofectamine for 6 h per 60-mm plate, according to the
instructions supplied. PRL in the vector used in stable transfectants,
pcDNAneo, was not produced well in transient transfections, so for
these experiments, PRL was used in pcDNA3 (Invitrogen, San Diego,
CA).
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FOOTNOTES
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Address requests for reprints to: Priscilla S. Dannies, Yale University School of Medicine, 333 Cedar Street, Department of Pharmacology, New Haven, Connecticut 06520-8066.
This work was supported by NIH Grant DK-46807.
Received for publication June 2, 1997.
Accepted for publication June 24, 1997.
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