From the Department of Pathology and Laboratory Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104
Received for publication, December 29, 2000, and in revised form, May 1, 2001
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ABSTRACT |
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The actions of prolactin (PRL) are mediated by
its receptor, a member of the superfamily of single transmembrane
cytokine receptors. High affinity binding proteins for the closely
related growth hormone have been found in the sera of several
species including humans and are generated by alternative splicing or proteolysis of the growth hormone receptor extracellular domain (ECD).
In contrast, no conclusive evidence has been presented that an
analogous prolactin-binding protein (PRLBP) is expressed in human
serum. Using both monoclonal and polyclonal antibodies generated
against hPRL and the ECD of the human prolactin receptor, co-immunoprecipitation analyses of human serum identified a 32-kDa hPRLBP capable of binding both hPRL and human growth hormone. A
measurable fraction of circulating PRL (36%) was associated with the
hPRLBP. Despite well documented sex differences in serum hPRL levels,
there were no significant differences in the levels of hPRLBP found in
the sera of normal adult males and females (15.3 ± 1.3 ng/ml
versus 13.4 ± 0.8 ng/ml, respectively (mean ± S.E.)). Immunoprecipitation studies also detected the PRLBP in human
milk albeit at lower concentrations than found in sera. Deglycosylation
did not alter its electrophoretic mobility, indicating an absence of
carbohydrate moieties and suggesting that the hPRLBP spans most of the
PRLR ECD, a result confirmed by limited proteolysis and mass
spectrometry. The potential function of this serum chaperone was
assessed in vitro by the addition of recombinant hPRLBP to the culture medium of the PRL-dependent Nb2 T-cell line.
These studies revealed that the hPRLBP antagonizes PRL action,
inhibiting PRL-driven growth in a dose-dependent manner.
Prolactin and growth hormone receptors are members of the cytokine
receptor superfamily. Both the prolactin receptor
(PRLR)1 and growth hormone
receptor (GHR) are single-chain transmembrane proteins composed of an
extracellular, transmembrane, and intracellular domain. The hormones
prolactin (PRL) and growth hormone (GH) exert their effects at the
molecular level by inducing the homodimerization of their respective
receptors, initiating the activation of receptor-associated kinases and
signaling cascades.
Until the last few decades, it was believed that peptide hormones
circulated freely, but experimental evidence has proven otherwise.
Although PRL was found to associate with serum IgG (1), a GH-binding
factor was initially identified in the serum of pregnant mice (2) and
subsequently discovered in rabbit (3) and human sera (4, 5). With the
subsequent cloning of the GHR cDNA, a GH-binding protein (GHBP) was
found to have amino acid sequence identity with the extracellular
domain of the membrane-bound GHR (6). The GHBP is a 246-residue
glycoprotein with a molecular mass of 50-60 kDa. The generation
of GHRBP occurs via two separate mechanisms in mammals. In the mouse
and rat, alternative splicing of a primary RNA transcript generates a
truncated receptor in which the transmembrane domain is replaced by a
short hydrophilic sequence (7-9). In other species, such as humans and
rabbits, a full-length GHR is expressed, but GHBP is generated by
proteolytic cleavage of the extracellular domain (6, 10, 11).
To date, although proteins in milk and serum have been found to
interact with PRL, the existence of a free PRLR extracellular domain
(ECD), i.e. a PRLBP, has not been established. Evidence for
PRL-binding proteins in milk has been shown within humans (12, 13) as
well as pigs, sheep, and rabbits (13, 14). Although the identity of two
serum PRL-binding proteins has been recently established, namely IgG
(1, 15) and cyclophilin B (16), no clear evidence exists for a PRLBP in
mammalian serum (14, 17).
In this study, we characterize a PRLBP in human serum with
identity to the ECD of the PRLR. The serum PRLBP was immunoreactive with both poly- and monoclonal antibodies generated against the extracellular domain of the PRLR and shows a proteolytic profile similar to a recombinant PRLR extracellular domain. Tryptic digest mass
spectrometry (MS) further confirmed identity with the PRLR ECD.
Coimmunoprecipitations revealed the association of the PRLBP with both
serum PRL and GH. Furthermore, unlike the GHBP, the serum PRLBP is not
glycosylated. Based on its PRL binding ability, it was also shown to
inhibit the hormone-induced proliferation of Nb2 cells.
Sera and Milk Samples--
Human sera were obtained from healthy
male and female adult donors. Aliquots were stored at Expression of Recombinant Human Prolactin-binding Protein
(rhPRLBP)--
A cDNA fragment of the human long PRLR was
amplified by polymerase chain reaction with primers homologous to the
mature form of the extracellular domain. The primers PRLR-1
(5'-CGAATTCCAGTTACCTCCTGGA-3') and PRLR-211'
(5'-GCTCGAGTCATGTATCATTCATGGT-3') were used in the 50-µl
amplification reaction with 50 ng of DNA template, 5 µl of 10 × polymerase chain reaction buffer, 3 µl of 25 mM
MgCl2, 1 µl of 10 mM dNTP mix, and 5 units of
Taq polymerase (Life Technologies, Inc.). After a
2-min incubation at 94 °C, the mixture was amplified for 30 cycles
of 94 °C for 30 s, at 47 °C for 30 s, and at 72 °C
for 30 s. It was then extended at 72 °C for 2 min. The
amplified fragment was purified and concentrated by phenol/chloroform
extraction followed by ethanol precipitation. The pellet was
resuspended in 40 µl of dH2O, and 10 µl of the
sample was digested with EcoRI and XhoI
restriction enzymes and ligated into the corresponding restriction
sites of pGEX4T-1 (Amersham Pharmacia Biotech). The clone was
subsequently checked for amplification errors by dideoxynucleotide sequencing. The resulting glutathione S-transferase
(GST)-ECD was expressed as per kit instructions. Briefly, a 1-liter
culture of Escherichia coli transformant was grown to
mid-log phase and induced with 0.1 mM
isopropyl-1-thio- Immunoprecipitation of PRLBP and PRL from Serum and
Milk--
Sera and milk samples (1 ml) were thoroughly precleared of
endogenous immunoglobulin by repeated overnight incubation with 300 µl of a protein A/G bead mixture. Precleared samples were then
incubated overnight at 4 °C with 5 µl of rabbit anti-PRLR antiserum developed by our laboratory and characterized elsewhere (19).
As a negative control, an equal aliquot of serum was incubated with 5 µl of normal rabbit serum. Immune complexes were then precipitated by
the addition of 50 µl of protein A beads for 30 min at 4 °C. After
three washes with lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1 mM
Na3VO4, and 10 µg/ml aprotinin, pepstatin,
and leupeptin), the beads were suspended in 30 µl of Laemmli buffer
with mercaptoethanol and boiled. For the immunoprecipitation of serum
PRL, samples were treated in a similar manner except that 5 µl of
goat anti-PRL antiserum (Santa Cruz Biotechnology, Inc., Santa Cruz,
CA) and 50 µl of protein G beads were used. For coimmunoprecipitation
of GH with PRLBP, samples were immunoprecipitated with 5 µl of rabbit
anti-PRLR antiserum plus 50 µl of protein A beads, washed three
times, and suspended in Laemmli buffer as stated above.
Immunoblot Analysis--
Immune complexes were resolved by
electrophoresis through a 12% SDS-polyacrylamide gel and transferred
to polyvinylidene difluoride membrane (Bio-Rad). Nonspecific
binding was blocked with 5% milk in PBS/Tween 20. Antigens were
labeled with 1:1000 dilutions of primary antibodies and 1:2500
dilutions of horseradish peroxidase-coupled secondary antibodies.
Antigen-antibody complexes were visualized by enhanced
chemiluminescence (Amersham Pharmacia Biotech). Anti-PRLR antiserum
used was described previously (19). Anti-PRLR monoclonal antibody
directed against the human PRLR ECD was the gift of Genzyme, Inc.
(Cambridge, MA). Rabbit anti-growth hormone antiserum was obtained from
the National Institute of Diabetes and Digestive and Kidney Diseases.
Quantitation of Serum PRLBP, PRL, and GH--
To assess the
quantities of PRLBP, PRL, and GH found in sera, immune complexes were
electrophoresed in parallel with known quantities of recombinant human
PRLBP, hPRL, or hGH. Visualized bands were then scanned, and signal
intensities were obtained using ImageQuant densitometry software
(Molecular Dynamics, Inc., Sunnyvale, CA). These data were used to
generate a concentration/intensity curve to quantitate unknown amounts
of PRLBP, PRL, or GH. Statistical analyses were performed using
GraphPad Prism version 3.00 for Windows (GraphPad
Software©, San Diego, California).
Deglycosylation of Serum PRLBP--
PRLBP was immunoprecipitated
overnight with 5 µl of anti-PRLR antiserum and 50 µl of protein A
beads as described above. Immune complexes were washed three times with
lysis buffer, followed by three washes with 50 mM sodium
phosphate, pH 7.5. Pellets were resuspended in 40 µl of 50 mM sodium phosphate with or without 10 units of
glycopeptidase F (Sigma) and incubated for 8 h at 37 °C.
Samples were washed once more with lysis buffer, resuspended in 40 µl
of Laemmli buffer, and boiled. The treated and untreated samples were
electrophoresed in parallel with 30 ng of recombinant PRLBP on a 12%
SDS-polyacrylamide gel. This served as a control for the
electrophoretic mobility of nonglycosylated PRLBP on a reducing gel.
Samples were transferred to polyvinylidene difluoride and blotted with
anti-PRLR mAb.
In-gel Protease Digestion of PRLBP--
Precleared serum (15 ml)
was immunoprecipitated with 15 µl of anti-PRLR antiserum as described
above, electrophoresed, and stained with Coomassie Brilliant Blue
(Bio-Rad). After extensive destaining with 45% methanol, 10% acetic
acid, the gel was washed with distilled water with several changes. The
bands corresponding to the PRLBP were excised with a razor blade and
suspended in 0.1 ml of 100 mM ammonium carbonate. Asp-N or
Lys-C protease (Sigma) was added (1:25 w/w), and the mixture was
thoroughly macerated with a microcentrifuge pestle (Kontes, Vineland,
NJ). After overnight digestion at 37 °C, peptide fragments were
separated from the gel pieces by centrifugation for 5 min at 2000 × g through a 1.5-ml low protein-binding microfilterfuge
tube. Tricine sample buffer was added to the collected samples in the
flow-through, boiled for 2 min, and separated on a 20%
Tris-Tricine polyacrylamide gel. Bands were visualized using the Silver
Stain Plus kit (Bio-Rad) per manufacturer's instructions and
analyzed using the Scion Image for Windows densitometry software
package (Scion, Inc., Frederick, MD).
Mass Spectrometry--
To isolate pure PRLBP from serum,
covalently coupled anti-PRLR beads were generated for
immunoprecipitation. Briefly, 200 µl of affinity-purified anti-PRLR
antiserum was coupled to 100 µl of protein A beads for 1 h at
room temperature. Beads were washed two times with 10 volumes of 0.2 M sodium borate (pH 9.0) and resuspended in 10 volumes of
0.2 M sodium borate, 20 mM dimethyl pimelimidate (pH 9.0) for 30 min at room temperature. Beads were washed
in 0.2 M ethanolamine (pH 8.0) and then incubated for
2 h at room temperature in 0.2 M ethanolamine. The
covalently coupled anti-PRLR beads were washed three times in PBS and
mixed with 10 ml of precleared human serum overnight at 4 °C. The
beads were then washed three times in 10 mM phosphate
buffer (pH 6.8), and bound protein was eluted in 5 M LiCl,
10 mM phosphate buffer (pH 7.2). Sample was mixed with
Laemmli buffer, boiled, and electrophoresed on a 10%
SDS-polyacrylamide gel. The gel was stained with Silver Stain Plus
(Bio-Rad), and the band corresponding to the PRLBP was excised for mass
spectrometry. Samples corresponding to a plain gel slice and rhPRLBP
were excised for use as negative and positive controls, respectively.
The gel slices were subjected to in-gel tryptic digestion by
rehydrating with 200 ng of sequencing grade trypsin (Promega, Madison,
WI). Extractable tryptic peptides were subjected to MALDI-MS analysis
using a paracrystalline film matrix method for desalting the extracts.
Monoisotopic mass lists for the samples were generated and submitted
for mass data base searching using the Profound algorithm (Rockefeller
University, New York, NY).
Inhibition of Nb2 Lymphoma Cell Proliferation with Recombinant
PRLBP--
Nb2-11C cells were maintained in Fisher's medium
supplemented with 10% FCS, 10% horse serum, 1 mM
L-glutamine, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 0.1 mM Expression of a PRLBP in Human Serum--
To date, no protein with
homology to the PRLR ECD has been found in sera, and the existence of
such a protein in milk has been only indirectly implicated (12-14).
This may in part be attributable to the absence of high avidity
anti-PRLR antibody commercially available. Using a recently
characterized anti-PRLR antiserum (19) in conjunction with a monoclonal
antibody, we revisited the existence of a bona fide PRLBP
(i.e. the free PRLR ECD) in human serum. To that end, human
serum extensively precleared of immunoglobulin was immunoprecipitated
with antiserum raised against the extracellular domain of the PRLR.
Immunoblot analysis of this precipitate with a specific anti-PRLR mAb
shows that the immunoprecipitate contained an immunoreactive protein
corresponding to the approximate molecular weight of the extracellular
domain of the human PRLR (Fig.
1A). This band was absent in
the control lane using normal rabbit serum for the immunoprecipitation,
illustrating the immunospecificity of the anti-PRLR antiserum. To
quantitate the amount of PRLBP in the donor serum, the
immunoprecipitation was repeated, and immunocomplexes were
electrophoresed in conjunction with known quantities of recombinant
PRLR (rPRLR) ECD (Fig. 1B). To ensure complete precipitation
of PRLBP, the serum was used in a second round of immunoprecipitation
(Fig. 1B, lane 3), revealing that the first
immunoprecipitation had concentrated nearly all of the serum PRLBP. By
comparison to the rPRLR ECD standards, it was determined that the donor
had 13.8 ± 1.4 ng (mean ± S.E.) of PRLBP/ml of serum.
The Serum PRLBP Binds Serum PRL and GH--
Unlike any other
species, hGH has the capacity to bind both the PRLR and GHR. Indeed,
previous studies have shown that a milk PRL-interacting protein and
GHBP are both capable of binding radiolabeled hGH (14). To determine
whether there is an association between hPRL or hGH and PRLBP in human
serum, coimmunoprecipitations were performed. As shown in the top
panel of Fig. 2A,
anti-PRLR immunoblot analysis of anti-PRL immunoprecipitates revealed
the association of the PRLBP with serum PRL. Stripping and reprobing
the blot with anti-PRL antibody shows that the immunoprecipitating
antiserum was specific for PRL (Fig. 2A, bottom
panel). As shown in Fig. 2B, anti-GH immunoblot
analysis of anti-PRLR immunoprecipitates revealed that hGH was also
bound to the PRLBP. To quantitate the amount of serum PRL complexed
with PRLBP, immunoprecipitations were performed using anti-PRLR and
anti-PRL antisera followed by immunoblotting with anti-PRL antiserum in
conjunction with known concentrations of a PRL standard. As shown in
Fig. 2C, 28 ng of PRL was found in 1 ml of a donor's serum,
of which 10 ng or 36% coimmunoprecipitated with PRLBP as determined by
known quantities of PRL electrophoresed in parallel. By similar
methods, the amount of serum GH coimmunoprecipitating with PRLBP was
analyzed. In this donor's serum, 2.6 ng of the 5 ng of GH
present in 1 ml of serum associated with PRLBP (data not shown). To
further investigate the PRL binding properties of the PRLBP, a
recombinant form of the PRLBP was generated in E. coli for
subsequent analysis by surface plasmon resonance. Using various
concentrations of rPRLBP, the dissociation constant
(Kd) of binding site I was measured at 13.4 nM, an observation similar to those reported in depth
elsewhere2 and in good
agreement with existing biosensor data (21).
Both Sexes Have Comparable Levels of Serum PRLBP--
Because the
previous experiments utilized the serum of a single, nonlactating
female donor, we wished to examine the variation in the levels of PRLBP
observed between the sexes. This was accomplished by anti-PRLR
immunoprecipitation analysis of donor serum and milk. As shown in Fig.
3, there was no significant difference in
the amounts of PRLBP found in the sera of nonpregnant, nonlactating females (lanes 1-5) and males (lanes
6-9) with respective levels averaging 15.3 ± 1.3 ng/ml versus 13.4 ± 0.8 ng/ml PRLBP. We also observed
no significant difference in the amounts of PRLBP found in the sera of
these women as compared with sera from 24-h postpartum mothers (data
not shown). Additionally, PRLBP was found in the breast milk of a
lactating mother (Fig. 3A, lane 10) although at
somewhat lower levels than in serum, as was similarly shown for other
mammals (13, 14).
Biochemical Identification of the PRLBP--
The PRLR is
known to be highly glycosylated on the asparagine residues of
its extracellular domain (22). To determine whether the PRLBP was
glycosylated to a similar degree, immunoprecipitated PRLBP was treated
with the general deglycosidase glycopeptidase F and then
electrophoresed along with recombinant PRLBP expressed in E. coli (Fig. 4A). No shift
in electrophoretic mobility was observed upon glycopeptidase F
treatment, suggesting that serum PRLBP was not glycosylated and had the
same apparent molecular mass as the recombinant form of the PRL
ECD (32 kDa). Although two antibodies generated against the
extracellular domain of the PRLR were immunoreactive against serum and
milk PRLBP (Figs. 1 and 2), we could not entirely rule out that this
was attributable to cross-reactivity to a highly homologous protein.
Attempts at N-terminal amino acid sequencing, however, were
noninformative, possibly because of a blocked N terminus. Therefore,
serum PRLBP was excised from an SDS-polyacrylamide gel and subjected to
protease digestion with Asp-N or Lys-C (Fig. 4B). As a
positive control, recombinant PRLR ECD was digested in parallel. GST
digestion served as a negative control. The peptide fragments resulting
from the digestion of serum PRLBP showed an electrophoretic profile
highly similar to the PRLR ECD (Fig. 4B, row 1 versus row 2). In contrast, the pattern differed
from that of GST (Fig. 4B, row 2 versus
row 3), suggesting that the PRLBP was indeed homologous to
the extracellular domain of the PRLR. To confirm this observation,
MALDI-MS was performed on PRLBP immunoprecipitated from serum as
compared with recombinant human PRLBP. Four tryptic peptides
with molecular masses of 971, 1399, 1772, and 1893 correlated
between the serum PRLBP and the positive control of rPRLR ECD (Fig.
4C, middle versus bottom
panel). Mass data base searching revealed homology to the PRLR
ECD, and two peaks corresponded to tryptic fragments of this region
(Fig. 4C, arrows in the bottom
panel).
Recombinant Human PRLBP Inhibits the Proliferation of Nb2 cells
Stimulated with Human PRL--
As the GHBP can modulate the activity
of GH, we wished to determine the effect of purified recombinant PRLBP
on the biological activity of hPRL using the rat Nb2-11C lymphoma cell
proliferation assay (Fig. 5). As
increasing concentrations of PRLBP were added, a 3-fold reduction in
cell proliferation was noted (Fig. 5A). Importantly, the
PRLBP by itself was not toxic to cells. Therefore, the overall decrease
in proliferation could be attributed to the PRLBP competing with cell
surface receptors for ligand. This competition also extended to other
members of the somatolactogenic family of ligands. As the control
protein BSA was observed to have no positive or negative effect on
PRL-induced proliferation (data not shown), we compared the effects of
coincubation of BSA or PRLBP with other somatolactogenic hormones on
Nb2 cell proliferation. The inclusion of PRLBP into the medium of
either PL- or GH-stimulated Nb2 cells was found to decrease
ligand-induced cell proliferation, an effect not observed for BSA (Fig.
5B).
Previous searches for the presence of a soluble form of the PRLR
in mammalian biological fluids only resulted in the discovery of a
putative PRLBP in milk samples (12-14). However, attempts to find a
PRLBP in sera were unsuccessful, resulting from the general lack of
robust anti-PRLR antibodies. In this study, we show evidence for a
soluble PRLBP in human plasma. Using a high avidity anti-PRLR antiserum
(19) generated by our laboratory, it was possible to immunoprecipitate
a protein from human plasma that was subsequently recognized by an
anti-PRLR monoclonal antibody during immunoblot analysis (Fig.
1A). This serum PRLBP was observed to have the same
molecular mass (32 kDa) as the extracellular domain of the human PRLR
expressed in E. coli and was not glycosylated based on the
inability of a glycosidase to affect its electrophoretic mobility (Fig.
4A). Our findings extend an observation by Mercado and
Baumann (12) in which a PRL-binding protein in milk bound to hGH
affinity columns, but unlike the GHBP in human plasma, did not bind to
wheat germ lectin columns, suggesting an absence of glycosylation.
However, this study was unable to confirm the immunological identity of
this species as that of the PRLR ECD. Interestingly, the predicted
molecular mass of rhPRLBP expressed in E. coli is 24.5 kDa,
yet both native and recombinant forms migrate as 32-kDa proteins. This
difference in the predicted and observed masses may be attributable to
the high content of acidic and proline residues comprising the PRLBP
(21%). Previous studies have reported this same discrepancy because of
the retarded mobility of a variety of proteins based on proline and
acidic residue content (23-25). To confirm the identity of the PRLBP
with the PRLR ECD, the immunoprecipitated PRLBP was subjected to
limited proteolysis and SDS-polyacrylamide gel electrophoresis analysis
alongside similarly digested rhPRLR ECD (Fig. 4B). Both
proteins exhibited similar peptide fingerprints from both digestions,
further supporting the correlation between the PRLBP and the
extracellular domain of the PRLR. Finally, MALDI-MS analysis and
monoisotopic mass lists for the sample revealed homology with the PRLR
by data base searching using the Profound algorithm. Although
there were four identical peptides between rhPRLR ECD and serum PRLBP,
two were found in the data base search to correspond to fragments of
the PRLR ECD. It is probable that incomplete tryptic digestion of the
samples would explain why the other two fragments were not identified
in the data base search. Thus, given the existing precedent with the
naming of the GHBP, we therefore have termed the serum protein
identified in these studies as the PRLBP.
The mechanism for PRLBP generation as well as its tissue source is
unknown. The generation of GHBP varies by species because rodents
produce GHBP by alternative splicing mechanisms (7, 8, 26), whereas
humans (6, 10) and rabbits (11) produce GHBP by the specific
proteolysis of the membrane receptor. Indeed, Saito et al.
(27) observed the constitutive release of GHBP from IM-9 cells. This
was believed to be the result of surface GHR being cleaved by a
metalloprotease because EDTA inhibited the production of GHBP.
Membrane-bound PRLR is known to be N-link glycosylated (28),
an event crucial for its cell surface targeting (22). The fact that a
soluble, nonglycosylated PRLBP is present in milk and serum in humans
therefore suggests that the binding protein may be produced by a
combination of deglycosylation and proteolysis of the full-length PRLR.
Indeed, although we observed the association of glycosylated
full-length PRLR from T47D cell lysates with concanavalin A beads,
neither rhPRLBP nor serum PRLBP was observed to bind to the beads (data
not shown). This suggests that only the deglycosylated form of the
PRLBP is found in serum. Interestingly, it has been shown that certain
plasma membrane glycoproteins of hepatocytes undergo rapid
deglycosylation (29, 30). As the liver abundantly expresses the PRLR
(31), proteolytic cleavage of deglycosylated hepatic receptors could be
one possible source of PRLBP in human serum. Current studies are
underway to address this hypothesis.
The PRLBP was present in the sera of both males and females at
comparable levels. This absence of sexual dimorphism is reminiscent of
the PRLR itself (32). Like previous reports, which indicated that a
protein capable of binding PRL was present in human milk (12, 14), our
data would now indicate that this species represents the PRLBP.
Additionally, the milk PRLBP was observed to be at a lower
concentration than serum PRLBP, which is consistent with the observed
levels of GHBP in human milk versus serum (12). Preliminary
data from our laboratory suggest that there is no significant increase
in serum PRLBP levels from mothers 24 h postpartum (data not
shown). Interestingly, this is in direct contrast with the levels of
GHBP observed during rodent gestation. In the mouse, increases in the
levels of serum GHBP are observable on Day 9 of gestation, and by late
gestation serum GHBP levels increase 30-fold (33). This may also be
regulated by GH itself because continuous exposure to elevated GH in
nonpregnant rats results in the up-regulation of GHBP (9, 34). In
contrast, however, it is interesting to note that a
pregnancy-associated rise in GHBP is not driven by GH (35).
Because the membrane-bound PRLR is capable of binding both PRL and GH,
we wished to determine the ligand binding characteristics of the PRLBP.
Surface plasmon resonance experiments for the hPRLBP were consistent
with previously reported hormone-receptor interactions such as those
observed by Gertler et al. (21). The PRLBP was found
associated with both PRL and GH in human serum, and ~36% of plasma
PRL was found associated with PRLBP in a single donor (Fig. 2). In a
separate single donor, ~53% of plasma GH was found to be
associated with the PRLBP. This result is similar to that observed with
GHBPs because GHBPs complex 45-55% of circulating GH under basal
conditions (36-38). This has been shown to slow the renal clearance of
GH, thereby providing a longer lasting reservoir of hormone (39, 40).
Quantitative analysis of the molar concentration of the PRLBP, GH, and
PRL indicated that the PRLBP is nearly saturated with somatolactogenic
hormone, suggesting that the ligand-bound PRLBP may serve as a
significant buffer to hormone levels.
Although a decrease in the clearance of PRL by the PRLBP could enhance
the activity of PRL in vivo, in vitro studies
using recombinant hGHBP have shown that GHBP inhibits GH activity (41). Similarly, the bovine PRLR extracellular domain has been shown to
inhibit the bioactivity of ovine PRL on Nb2 cells (42). Because the hPRLBP was capable of binding PRL, we wished to determine whether a
recombinant form of the human PRLBP could inhibit the hPRL-induced
proliferation of Nb2 cells (Fig. 5A). A
dose-dependent decrease in Nb2 cell proliferation was
observed when recombinant hPRLBP was added to cells. This was also
observed in the presence of hPL and hGH (Fig. 5B),
suggesting that the PRLBP is capable of binding these hormones as well.
Although PRLBP was capable of inhibiting PRL-mediated cell
proliferation in vitro, the effects of PRLBP in
vivo may differ greatly. In a rat model, human GHBP complexed with
hGH was cleared at a significantly slower rate, exhibited limited
extravascular availability, and degraded at a slower rate than free hGH
(39). Similarly, hypophysectomized rats given GH in conjunction with
GHBP showed significantly greater growth and weight gain compared with
animals given GH alone (20). Based on these findings, the
in vivo presence of PRLBP may serve to maintain a reservoir
of PRL in the circulation, providing a releasable pool of free hormone
in times of diminishing supply.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C and
clarified by centrifugation at 12,000 × g for 30 min
before use. Human milk was obtained from a lactating mother and
defatted by centrifugation. Infranatant was collected by avoiding the
upper lipid layer as well as any sediment and stored at
80 °C.
-D-galactopyranoside for 4 h. Pelleted cells were suspended in 20 ml of 10 mM EDTA and 10 mM Tris-HCl, pH 8.0, in the presence of 0.5 mg/ml
lysozyme. Purification and solubilization of refolded GST-ECD was then
carried out as described previously (18). For purification of
bioreactive recombinant human PRLBP (rhPRLBP), 1 ml of refolded GST-ECD
preparation was incubated with 300 µl of glutathione beads for 30 min
at 4 °C. After washing the beads three times with PBS, protein was eluted in 300 µl of PBS containing 10 mM reduced
glutathione. Thrombin protease (10 units) (Amersham Pharmacia Biotech)
was added to the elution at room temperature for 18 h. The
digested protein solution was dialyzed overnight in 4 liters of PBS and cleared of GST protein by three incubations with fresh glutathione beads. The purified rhPRLBP was electrophoresed on a 12%
SDS-polyacrylamide gel and stained with Coomassie Blue to ensure the
complete removal of GST protein as well as undigested fusion protein.
The sample was incubated for 1 h with polymyxin beads to remove
residual lipopolysaccharide and then filter-sterilized, aliquoted, and flash frozen.
-mercaptoethanol. To assess the
ability of recombinant hPRLBP to inhibit PRL-induced cellular
proliferation, 1 × 104 Nb2 cells were aliquoted in
triplicate wells in medium consisting of Dulbecco's modified Eagle's
medium (Life Technologies, Inc.) supplemented with sodium selenide,
linoleic acid, insulin, and transferrin (ITS+; Calbiochem) in the
presence or absence of 50 pM hPRL, hPL, or hGH. Increasing
concentrations (0-100 nM) of recombinant hPRLBP or 100 nM hPL, hGH, or BSA were simultaneously added to the wells.
After overnight culture, cells were pulsed with 1 µCi of
[3H]thymidine at 37 °C for 4 h. The incorporation
of radiolabel was determined by scintillography of the harvested,
washed cells.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
PRLBP is expressed in human serum.
A, immunoprecipitation of PRLBP from serum. Samples (1 ml)
of precleared serum were immunoprecipitated in 5 µl of normal rabbit
serum (NRS) or anti-PRLR antiserum overnight at 4 °C.
After washing, precipitates were analyzed by immunoblot using anti-PRLR
mAb (1:1000). An E. coli-expressed fusion of the hPRLR ECD
to GST (GST-ECD) served as a positive control for antibody specificity.
These results are representative of one of three experiments.
B, quantitation of PRLBP in human serum. PRLBP was
immunoprecipitated from serum as in panel A. To ensure the
complete precipitation of PRLBP, serum was immunoprecipitated a second
time (2° -PRLR). Immunoprecipitates were electrophoresed in
parallel with known quantities of recombinant PRLBP. Amounts of PRLBP
in serum were calculated based on densitometry quantitation of
standards. These results are representative of one of three
experiments.
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Fig. 2.
PRLBP associates with PRL and GH in human
serum. A, association of serum PRL with PRLBP.
Precleared serum was immunoprecipitated with normal rabbit serum
(NRS) or goat anti-PRL antiserum
( -PRL). Immunoprecipitates were subjected to
immunoblot analysis with
-PRLR mAb (top panel). The blot
was stripped and reprobed with a 1:1000 dilution of
-PRL antiserum
to ensure the immunoreactive specificity of the antiserum (bottom
panel). These results are representative of one of three
experiments. B, association of serum GH with PRLBP.
Precleared serum was immunoprecipitated with normal rabbit serum or
anti-PRLR antiserum (
-PRLR), and
immunoprecipitates were subjected to immunoblot analysis with a 1:1000
dilution of
-GH antiserum (top panel). The blot was
stripped and reprobed with a 1:1000 dilution of
-PRLR mAb
(bottom panel). These results are representative of one of
two experiments. C, quantitation of PRL bound to PRLBP in
serum. Precleared serum samples (1 ml) were immunoprecipitated
overnight with anti-PRLR, anti-PRL, or normal rabbit serum.
Immunoprecipitates were electrophoresed in conjunction with known
quantities of recombinant hPRL to determine the percentage of serum PRL
complexed with PRLBP. These results are representative of one of two
experiments.
View larger version (23K):
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Fig. 3.
PRLBP is expressed in the sera of both males
and females. A, immunoblot of serum samples. Precleared
sera samples and breast milk were immunoprecipitated with -PRLR
antiserum and subjected to immunoblot analysis with a 1:1000 dilution
of anti-PRLR. B, quantitation of PRLBP in the sera of both
sexes. Bands from panel A were analyzed by
densitometry against known quantities of recombinant PRLBP to
quantitate the amounts of PRLBP found in serum. Males, 15.3 ± 1.3 ng/ml; females, 13.4 ± 0.8 ng/ml (mean ± S.E.).
NRS, normal rabbit serum.
View larger version (29K):
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Fig. 4.
Biochemical characterization of the
PRLBP. A, PRLBP is not glycosylated. Precleared serum
was immunoprecipitated with anti-PRLR antiserum, and the
immunoprecipitate was washed and incubated with or without 10 units of
glycopeptidase F (PNGase F) in buffer for 8 h
at 37 °C. Samples were immunoblotted in parallel with recombinant
PRLBP and probed with a 1:1000 dilution of anti-PRLR mAb. B,
proteolytic analysis of serum PRLBP versus PRLR ECD. Serum
was immunoprecipitated with anti-PRLR antiserum as described above,
electrophoresed, and stained with Coomassie Brilliant Blue. Excised
bands were digested with Asp-N and Lys-C, and peptide fragments were
separated on a 20% Tris-Tricine polyacrylamide gel. Bands were
visualized using the Silver Stain Plus kit (Bio-Rad) and scanned for
densitometric analysis. Recombinant PRLR ECD served as a positive
control, and GST served as a negative control. These results are
representative of one of two experiments. C, MALDI-MS
identification of the hPRLBP. Serum was immunoprecipitated with protein
A beads covalently linked to anti-PRLR antibodies. Eluted
hPRLBP was electrophoresed and stained with Silver Stain Plus
(Bio-Rad). Excised bands were digested with trypsin and subjected to
MALDI-MS. Two peptides corresponding to the ECD of the human PRLR are
indicated. rECD, recombinant ECD; pT, trypsin
autolysis product; TOF, time of flight; LD, limit
of detection.
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Fig. 5.
Effect of rhPRLBP on the proliferation of Nb2
lymphoma cells. A, rhPRLBP blocks hPRL-induced Nb2 cell
proliferation in a dose-dependent manner. 1 × 104 cells were plated overnight in Dulbecco's modified
Eagle's medium/ITS+ supplemented with 50 pM hPRL and
increasing concentrations of hPRLBP. Proliferation was assessed by
tritiated thymidine uptake. Results are mean
disintegrations/min (DPM) of triplicate wells ± S.E. B, rhPRLBP blocks Nb2 cell proliferation induced by
hPL and hGH. 1 × 104 cells were plated overnight in
Dulbecco's modified Eagle's medium/ITS+ supplemented with 50 pM hPRL, hPL, or hGH in conjunction with 100 nM
rhPRLBP or 100 nM BSA. Proliferation was assessed by
tritiated thymidine uptake. Results are mean disintegrations/min of
triplicate wells ± S.E.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank David Goodman and Anne Crivaro for providing us with human sera samples. We thank William Moore for contributing to the MALDI-MS analysis.
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FOOTNOTES |
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* This study was supported in part by Grants 2R01CA69294 (to C. V. C.) and 1F32DK09727 (to J. B. K.) from the National Institutes of Health.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.
To whom correspondence should be addressed: Dept. of Pathology and
Laboratory Medicine, University of Pennsylvania Medical Center, 513 Stellar-Chance Laboratories, 422 Curie Blvd., Philadelphia, PA
19104. E-mail: clevengc@mail.med.upenn.edu.
Published, JBC Papers in Press, May 3, 2001, DOI 10.1074/jbc.M011786200
2 J. B. Kline and C. V. Clevenger, submitted for publication.
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ABBREVIATIONS |
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The abbreviations used are: PRLR, prolactin receptor; PRL, prolactin; GH, growth hormone; GHR, growth hormone receptor; PRLBP, prolactin-binding protein; GHBP, growth hormone-binding protein; h, human; rh, recombinant human; r, recombinant; PL, placental lactogen; GST, glutathione S-transferase; ECD, extracellular domain; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MALDI-MS, matrix-assisted laser desorption/ionization-mass spectrometry; PBS, phosphate-buffered saline; BSA, bovine serum albumin; mAb, monoclonal antibody.
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REFERENCES |
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