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© The Histochemical Society, Inc.
0022-1554/98/$3.30
737
ARTICL
E
Volume 46(6): 737–743, 1998
The Journal of Histochemistry & Cytochemistry
http://www.jhc.org
Placental Lactogen-I (PL-I) Target Tissues Identified with an
Alkaline Phosphatase–PL-I Fusion Protein
Heiner Müller,
1
Guoli Dai, and Michael J. Soares
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas
SUMMARY
The rat placenta expresses a family of genes related to prolactin (PRL). Target
tissues and physiological roles for many members of the PRL family have yet to be deter-
mined. In this investigation we evaluated the use of an alkaline phosphatase (AP) tag for
monitoring the behavior of a prototypical member of the PRL family, placental lactogen-I
(PL-I). A probe was generated consisting of a fusion protein of human placental AP and rat
PL-I (AP–PL-I). The AP–PL-I construct was stably expressed in 293 human fetal kidney cells, as
was the unmodified AP vector that served as a control. AP activity was monitored with a
colorimetric assay in conditioned medium from transfected cells. Immunoreactivity and
PRL-like biological activities of the AP–PL-I fusion protein were demonstrated by immuno-
blotting and the Nb2 lymphoma cell proliferation assay, respectively. AP–PL-I specifically
bound to tissue sections known to express the PRL receptor, including the ovary, liver, and
choroid plexus. Binding of AP–PL-I to tissues was specific and could be competed with ovine
PRL. The results indicate that AP is an effective tag for monitoring the behavior of PL-I and
suggest that this labeling system may also be useful for monitoring the actions of other
members of the PRL family.
(J Histochem Cytochem 46:737–743, 1998)
T
he prolactin
(PRL)
gene family
contains at least
10 members some of which are expressed in the ante-
rior pituitary, placenta, and/or uterus (Soares et al.
1998). Within this gene family, information is avail-
able on target cells and biological actions for only a
subset of members utilizing the PRL receptor (classical
members) and those regulating angiogenesis (Jackson
et al. 1994; Volpert et al. 1996).
An important first step in understanding the actions
of a hormone/cytokine is to identify its targets. Typi-
cally, target cells for various hormones/cytokines have
been studied by radiolabeled ligand autoradiography or,
when reagents are available to the designated ligand
receptor, by immunocytochemical or in situ hybridiza-
tion procedures. The former requires the isolation of a
biologically active ligand to homogeneity and the lat-
ter requires identification and characterization of the
receptor system used by the ligand. Neither of these
options is readily available for nonclassical members
of the PRL family whose biological actions during
pregnancy are poorly understood. Flanagan and co-
workers developed an alternative approach, involving
the generation of alkaline phosphatase (AP)–ligand fu-
sion proteins, that has proved particularly useful for
identifying components of receptor tyrosine kinase
signaling pathways, including ligands and receptors
(Flanagan and Leder 1990; Cheng and Flanagan 1994;
Cheng et al. 1995; Chiang and Flanagan 1995,1996).
In this study, we have determined the effectiveness
of utilizing an AP tag to monitor a prototypical mem-
ber of the PRL family, placental lactogen-I (PL-I). PL-I
is a glycoprotein, as are most members of the PRL
family, and is secreted by trophoblast giant cells of the
developing placenta from immediately postimplanta-
tion until midgestation (Faria et al. 1990). The actions
of PL-I have been primarily studied via the generation
of recombinant PL-I in heterologous cell types (Colosi
et al. 1988; Robertson et al. 1994; Dai et al. 1996).
PL-I has been shown to bind PRL receptors (MacLeod
et al. 1989; Freemark et al. 1993) and possesses a
Correspondence to: Dr. Michael J. Soares, Dept. of Molecular
and Integrative Physiology, U. of Kansas Medical Center, Kansas
City, KS 66160–7401.
1
Present address: Department of Obstetrics and Gynecology,
University of Rostock, Rostock, Germany.
Received for publication September 3, 1997; accepted January
14, 1998 (7A4458).
KEY WORDS
alkaline phosphatase fusion
protein
ovary, corpus luteum prolactin
receptors
liver, hepatic prolactin
receptors
Nb2 lymphoma cells
placental lactogen-I
pregnancy
prolactin receptor
738
Müller, Dai, Soares
number of actions previously attributed to pituitary
PRL (Soares et al. 1998).
Collectively, the studies presented here indicate that
the AP labeling system is an effective means of moni-
toring interactions of PL-I with its target cells and sug-
gest the potential use of this labeling system for deter-
mining target cells for other PRL family members.
Materials and Methods
Reagents
Fetal bovine serum (FBS) and donor horse serum (HS) were
purchased from Sigma (St Louis, MO). Reagents for poly-
acrylamide gel electrophoresis were purchased from Bio-Rad
(Hercules, CA). All restriction enzymes, polymerases, and
DNA ligase were purchased from New England Biolabs
(Beverly, MA). The 293 cell line of human fetal kidney ori-
gin was obtained from American Type Culture Collection
(Rockville, MD). Transformation competent Sure bacterial
cells and random primer labeling kits were acquired from
Stratagene (La Jolla, CA). DNA extraction kits were pur-
chased from Qiagen (Chatsworth, CA). Nitrocellulose was
obtained from Schleicher & Schuell (Keene, NH). Ovine
PRL was purchased from Nobl Laboratories (Sioux City,
IA). T7 DNA sequencing kits were acquired from United
States Biochemical (Cleveland, OH). The pCMV/SEAP vec-
tor was acquired from Tropix (Bedford, MA). Radiolabeled
nucleotides were purchased from DuPont–NEN (Boston,
MA). Reagents for detection of immune complexes by en-
hanced chemiluminescence were acquired from Amersham
(Arlington Heights, IL). Unless otherwise noted, all other
chemicals and reagents were purchased from Sigma.
Animals and Tissue Preparation
Holtzman rats were obtained from Harlan Sprague–Dawley
(Indianapolis, IN). The animals were housed in an environ-
mentally controlled facility, with lights on from 0600 to
2000 hr, and were allowed free access to food and water.
Timed pregnancies and tissue dissections were performed as
previously described (Soares et al. 1985; Faria et al. 1990).
The presence of a copulatory plug or sperm in the vaginal
smear was designated Day 0 of pregnancy. Protocols for the
care and use of animals were approved by the University of
Kansas Animal Care and Use Committee.
Generation of the AP–PL-I Fusion Protein
Construction of the AP–PL-I Vector.
A fusion protein con-
sisting of a modified human placental AP (PLAP) and rat PL-I
was generated and used to monitor PL-I target cell interactions.
PLAP, in its native form, is a heat-stable, membrane-associ-
ated AP. The carboxy terminus of PLAP mediates membrane
binding. Using oligodeoxynucleotide-directed mutagenesis,
Berger and co-workers (1988) engineered a carboxy-termi-
nal truncation that resulted in a secreted PLAP referred to as
SEAP. The coding sequence for SEAP was subsequently lo-
calized downstream of the CMV promoter in a vector con-
taining ampicillin and neomycin resistance genes (pCMV/
SEAP; Tropix). A nucleotide region representing the carboxy
terminal 197 amino acids of the mature rat PL-I protein was
amplified (Dai et al. 1996) and ligated into the pCMV/SEAP
vector. Ligation with the PL-I insert resulted in a CMV pro-
moter-driven vector containing the ligated cDNAs encoding
a SEAP-PL-I fusion protein (AP–PL-I; see Figure 1). DNA se-
quencing of the insert was performed to verify the accuracy
of the PCR amplification.
Transfection, Selection, and Cloning.
After linearization with
Bgl II, the AP–PL-I construct was electroporated into 293
cells. After a 2-week selection with 500
m
g/ml G418, single
clones were isolated by limiting dilution and screened for AP
expression. An unmodified pCMV-SEAP vector (AP) was
similarly transfected, and selected, and served as a negative
control.
Preparation and Characterization of Medium Condi-
tioned by the AP and AP–PL-I-transfected 293 Cells.
Transfected 293 cells were cultured in MEM medium sup-
plemented with 20 mM HEPES, 100 U/ml penicillin, 100
m
g/
ml streptomycin, and 10% FBS in an atmosphere of 5%
CO
2
/95% air at 37C in a humidified incubator. After the
cells reached confluence, the medium was changed to serum-
free MEM
1
HEPES, further conditioned for 72 hr, col-
lected and clarified by centrifugation, sterile-filtered (0.22
m
m), and stored at
2
20C until used. AP activity was mea-
sured from conditioned medium via a colorimetric assay.
Initially, samples were heated for 30 min in a 65C waterbath
to inactivate endogenous heat-labile APs. Samples were then
incubated at room temperature (RT) in a glycine buffer (50
Figure 1 Construction of the AP–PL-I expression vector. A fusion
protein consisting of a modified human placental AP (PLAP) and
rat PL-I was generated and used to monitor PL-I target cell interac-
tions. PLAP in its native form is a heat-stable, membrane-associated
AP. The carboxy terminus of PLAP was truncated, resulting in a se-
creted PLAP referred to as SEAP and situated downstream of the
CMV promoter in a vector containing ampicillin and neomycin re-
sistance genes (pCMV/SEAP). A nucleotide region representing the
carboxy-terminal 197 amino acids of the mature rat PL-I was ampli-
fied and ligated into the pCMV/SEAP vector. Ligation with the PL-I
insert resulted in a CMV promoter-driven vector containing the li-
gated cDNAs encoding a SEAP-PL-I fusion protein (AP–PL-I).
PL-I Target Cells
739
mM glycine, pH 10.4, 0.5 mM MgCl
2
, 0.5 mM ZnCl
2
) con-
taining nitrophenylphosphate (0.5 mg/ml) in a total reaction
volume of 200
m
l. After a 5-min incubation absorbance was
measured at 405 nm. One unit of AP is defined as the
amount of enzyme that hydrolyzes 1
m
mole of
p
-nitrophe-
nylphosphate to
p
-nitrophenol in 1 min at 37C in a volume
of 1 ml.
Western Blot Analysis of PL-I
Western blot analysis was performed as previously described
(Hamlin et al. 1994; Dai et al. 1996). AP–PL-I preparations
were isolated from conditioned medium using immunopre-
cipitation with monoclonal antibodies to AP conjugated to
agarose (Sigma). Samples were washed, eluted from agarose
with treatment buffer, and separated by polyacrylamide gel
electrophoresis in 7.5% gels under reducing conditions.
Concentrated conditioned medium (
3
10) from differenti-
ated Rcho-1 trophoblast cells served as a positive control for
PL-I (Dai et al. 1996). Proteins from the gels were electro-
phoretically transferred to nitrocellulose. Polyclonal anti-
bodies generated against a synthetic peptide corresponding
to amino acids 1–19 of the mature PL-I protein (Hamlin et
al. 1994) were used as probes. Immune complexes were de-
tected using the enhanced chemiluminescence system as pre-
viously described (Dai et al. 1996).
Biological Characterization of AP–PL-I
PRL-like biological activities were assessed by use of the rat
Nb2 lymphoma cell proliferation assay (Tanaka et al. 1980;
Cohick et al. 1996). Nb2 lymphoma cells were routinely grown
in Fischer’s medium supplemented with 50
m
M 2-mercapto-
ethanol, 100 U/ml penicillin, 100
m
g/ml streptomycin, and
containing both 10% HS and 10% FBS (maintenance me-
dium
5
MM) in an atmosphere of 5% CO
2
/95% air at 37C.
Twenty-four hours before initiation of the assay, cells were
harvested, washed with the supplemented Fischer’s medium
containing only 10% HS (stationary medium
5
SM), and di-
luted to a concentration of 100,000 cells/ml. At the initiation
of the assay, cells were washed and aliquotted into 16-mm wells
(100,000 cells/ml/well) of a 24-well culture plate. Ovine PRL,
AP, or AP–PL-I preparations were added at various concen-
trations and the cells incubated for an additional 72 hr. Sam-
ples of treated cells were collected and counted in a Sysmex
Microcell counter (Model CC-110; TOA Medical Electron-
ics, Tokyo, Japan). Treatments were performed in triplicate.
In Situ Analysis of AP–PL-I Binding to Rat Tissues
Tissues were frozen in liquid nitrogen and stored at
2
70C
until tissue sections were prepared with the aid of a cryostat.
Sections were mounted onto glass slides, washed in a modi-
fied Hank’s balanced salt solution (HBHA; containing 20
mM HEPES, 0.5 mg/ml BSA, and 0.1% NaN
3
), and incu-
bated with AP, AP–PL-I, or AP–PL-I
1
excess PRL for 75
min at RT. After incubation, the sections were washed with
HBHA supplemented with 0.1% Tween 20 and fixed for 2
min in a 20 mM HEPES buffer containing acetone (60%)
and formaldehyde (3%). The fixed sections were washed,
heated at 65C for 30 min to inactivate endogenous tissue AP
activity, and then processed for detection of the heat-stable
AP activity associated with the fusion proteins, and cover-
slips mounted in aqueous mounting medium. The AP and AP
fusion protein were used at a concentration of 450 mU/ml.
The specificity of binding was assessed by the addition of ovine
PRL (5
m
g/ml) to some of the tissue section incubations.
Statistical Analysis
Data were analyzed by one-way ANOVA. The source of
variation from significant F ratios was determined with Stu-
dent’s two-tailed
t
-test (Keppel 1973).
Results
As an important step towards demonstrating the suit-
ability of the AP labeling system for monitoring activi-
ties of members of the PRL family, we generated and
characterized an AP–PL-I fusion protein. The con-
struction of the AP–PL-I vector was achieved by in-
frame insertion of the cDNA sequence of mature rat
PL-I downstream of the SEAP coding sequence within
the pCMV-SEAP vector (Figure 1).
Generation and Characterization of the AP–PL-I
Fusion Protein
The AP-PL-I construct and an unmodified AP control
vector were transfected via electroporation into 293
Figure 2 Alkaline phosphatase activity in conditioned medium
from transfected and nontransfected cells. The AP–PL-I construct
and an AP control construct were transfected via electroporation
into 293 cells. After selection with G418, the highest expressing
clones were identified and expanded. Transfected cells and non-
transfected controls were seeded at 100,000 cells/ml into 24-well
plates and cultured in MEM 1 HEPES 1 10% FBS. After 24 hr, the
cells were either washed with HBSS and the medium changed to
serum-free MEM (SFW), serum-free MEM without washes (SF),
MEM 1 3% FBS (3% FBS), or to MEM 1 10% FBS (10% FBS). After
72-hr incubation, conditioned medium was collected and heat-
in-activated for 30 min in a 65C waterbath. Absorbance at 405 nm
was measured after a 5-min incubation at room temperature of
the sample with the nitrophenylphosphate substrate.
740
Müller, Dai, Soares
cells. The presence of AP activity in conditioned me-
dium from transfected cells and nontransfected cells
was evaluated using a colorimetric assay. Several con-
ditions for the production of recombinant protein were
evaluated (Figure 2). Heat-stable AP activity secreted
by AP- and AP–PL-I-transfected 293 cells was enhanced
by the presence of FBS in the culture medium. The ele-
vated AP activity in serum-containing cultures likely
reflected stronger CMV promoter activity or possibly
decreased protein degradation. Therefore, the genera-
tion of the AP–PL-I fusion protein did not interfere
with AP enzymatic activity.
Anti-PL-I antibodies specifically recognized the AP–
PL-I fusion protein. AP and the AP–PL-I fusion pro-
tein were initially enriched by immunoprecipitation
with a monoclonal antibody to human PLAP conju-
gated to agarose. As shown by Western blot analysis,
PL-I antibodies recognized an AP–PL-I protein species
approximating 110 kD and native PL-I protein species
synthesized by differentiated Rcho-1 trophoblast cells
with sizes ranging from 36 to 45 kD (Figure 3). The
Figure 3 Western blot analysis of AP–PL-I. Samples were sepa-
rated by polyacrylamide gel electrophoresis in 7.5% gels under re-
ducing conditions and were electrophoretically transferred to ni-
trocellulose. Polyclonal antibodies generated against a synthetic
peptide corresponding to amino acids 1–19 of the mature PL-I were
used as probes. Immune complexes were detected using the en-
hanced chemiluminescence system. Lane A, AP–PL-I preparations
were isolated from conditioned medium using immunoprecipita-
tion with monoclonal antibodies to AP conjugated to agarose.
Lane B, Concentrated conditioned medium (310) from differenti-
ated Rcho-1 trophoblast cells served as a positive control for PL-I.
The AP–PL-I fusion protein migrated at an M
r
approximating 110
kD, and trophoblast cell-produced PL-I migrated at M
r
ranging
from 36 to 45 kD.
Figure 4 Effects of AP–PL-I on rat Nb2 lymphoma cell prolifera-
tion. PRL-like biological activities were assessed by use of the rat
Nb2 lymphoma cell proliferation assay. Nb2 lymphoma cells were
grown in supplemented Fischer’s medium containing 10% HS and
10% FBS (maintenance medium 5 MM). Twenty-four hours before
initiation of the assay, cells were harvested, washed with the sup-
plemented Fischer’s medium containing only 10% HS (stationary
medium 5 SM), and diluted to a concentration of 100,000 cells/ml.
At the initiation of the assay, cells were washed and aliquotted
into 16-mm wells (100,000 cells/ml/well) of a 24-well culture plate.
Ovine PRL, AP, or AP–PL-I preparations were added at various con-
centrations and the cells incubated for an additional 72 hr and
then counted. The AP and AP–PL-I preparations represent condi-
tioned medium from 293 cells and are calibrated on the basis of AP
activity. One unit of AP is defined as the amount of enzyme that
hydrolyzes 1 mmole of p-nitrophenyl-phosphate to p-nitrophenol
in 1 min at 37C in a volume of 1 ml. Treatments were performed
in triplicate. Results are presented as mean 6 SD. The PRL- and
AP–PL-I-treated cultures significantly stimulated the proliferation
of the Nb2 lymphoma cells compared to control cells in SM
(*p,0.001). n.s., not significant.
110-kD M
r
of the AP–PL-I fusion protein was consis-
tent with the predicted M
r
of its AP and PL-I compo-
nents. The AP control preparation was not recognized
by the PL-I antibodies (data not shown).
Biological Characterization of AP–PL-I
For the AP–PL-I fusion protein to be a useful probe
for monitoring PL-I interactions with its target tissues,
the AP–PL-I should be able to mimic PRL biological
actions. This issue was addressed by examining the ac-
tions of the AP–PL-I fusion protein on rat Nb2 lym-
phoma cells. The AP–PL-I fusion protein was capable
of significantly stimulating the proliferation of rat
Nb2 lymphoma cells in a concentration-dependent
manner (Figure 4). Rat Nb2 lymphoma cell prolifera-
tion is dependent on activation of the PRL receptor
signaling pathway, which can be achieved by PRL or
Figure 5
In situ analysis of AP-PL-I binding to rat tissues. Tissues sections were prepared with the aid of a cryostat. Sections were mounted
on glass slides, washed, and incubated with AP (
A,D,G
), AP–PL-I (
B,E,H
), or AP–PL-I
1
PRL (
C,F,I
) for 75 min at room temperature. After incu-
bation the sections were washed, fixed, washed, heated at 65C for 30 min to inactivate endogenous tissue AP activity, and processed for de-
tection of the heat-stable AP activity. The specificity of binding was further assessed by the addition of ovine PRL (5
m
g/ml) to some of the
tissue section incubations (
C,F,I
). (
A–C
) Ovary from Day 9 of pregnancy. (
D–F
) Frontal section of choroid plexus from female rat brain. (
G–I
)
Liver from Day 9 of pregnancy. Bars
5
100
m
m.
PL-I Target Cells
741
742
Müller, Dai, Soares
other ligands capable of interacting with the PRL re-
ceptor, such as PL-I (Tanaka et al. 1980; Colosi et al.
1988; Robertson et al. 1994; Cohick et al. 1996; Dai
et al. 1996) (Figure 4). The AP control did not signifi-
cantly stimulate rat Nb2 lymphoma cell proliferation
(Figure 4).
Because the AP–PL-I fusion protein retained the
ability to bind and activate PRL receptor signaling sys-
tems in vitro, we next evaluated its utility as an in situ
probe for identifying PL-I target cells within tissue sec-
tions. AP, AP–PL-I, or AP–PL-I
1
excess ovine PRL
were incubated with sections from several tissues, in-
cluding ovaries from midgestation rats, liver tissues
from midgestation rats, and brains from normal fe-
male rats (Figure 5). AP failed to demonstrate signifi-
cant binding to any structures in the tissues investi-
gated. AP–PL-I showed cell type-specific patterns of
binding to the tissue sections that could be competed
with excess ovine PRL (Figure 5). In the ovary, AP–
PL-I was specifically localized to the corpus luteum,
and binding within the brain was most prominent in
the choroid plexus. AP–PL-I binding to liver sections
was relatively homogeneous. On the basis of present
evidence, the AP–PL-I fusion protein appears to be a
useful probe for monitoring PL-I interactions with its
target tissues and the localization of PRL receptors.
Discussion
In this report we have described the generation and
characterization of an AP–PL-I fusion protein. The fu-
sion protein was generated by stably expressing a fu-
sion gene containing the heat-stable human placental
AP cDNA upstream of the rat PL-I cDNA in the 293
human fetal kidney cell line. The AP–PL-I fusion pro-
tein retained AP enzymatic activity, PL-I immunologi-
cal and biological activities, and represents an impor-
tant advance in the generation of a probe for monitoring
PL-I interactions with its target cells. Therefore, in ad-
dition to investigating growth factor receptor–tyrosine
kinase interactions (Flanagan and Leder 1990; Cheng
and Flanagan 1994; Cheng et al. 1995; Chiang and
Flanagan 1995,1996) and leptin–leptin receptor inter-
actions (Tartaglia et al. 1995), the AP tag system also
appears suitable for investigating the interactions of a
class of ligands belonging to the PRL family with their
receptors.
PL-I is a PRL receptor agonist expressed from im-
plantation until midgestation by trophoblast giant cells
situated at the maternal–placental interface (Colosi et
al. 1988; MacLeod et al. 1989; Faria et al. 1990;
Freemark et al. 1993; Robertson et al. 1994; Dai et al.
1996; Soares et al. 1998). Recombinant PL-I has pre-
viously been shown to bind specifically to liver and
ovarian membrane preparations (MacLeod et al. 1989),
most likely via specific interactions with the extracel-
lular domain of the PRL receptor (Sakal et al. 1996).
In situ analyses with radiolabeled recombinant PL-I
have localized PL-I target cells to the decidua of the
midgestation conceptus (Freemark et al. 1993) and to
the hypothalamus and choroid plexus of the pregnant
rat (Pihoker et al. 1993). We have similarly localized
AP–PL-I binding to the maternal liver, choroid plexus,
and corpus luteum of ovaries from pregnant rats. All
of the in vitro and in situ analyses indicate that PL-I
interacts with its target cells via PRL receptors. The
interactions of PL-I with luteal cells is consistent with
the actions of PL-I as a luteotropin (Galosy and Tala-
mantes 1995) and the distribution of PRL receptor
mRNAs in the corpus luteum during pregnancy (Clarke
and Linzer 1993; Ouhtit et al. 1993). The complete
distribution of PL-I tissue interactions in maternal, ex-
traembryonic, and extraembryonic compartments dur-
ing midgestation is yet to be resolved. In addition, it is
not clear whether PL-I interacts with all PRL receptors
or a subset of PRL receptors, and whether the activa-
tion of the PRL receptor signaling pathway in various
target cells is identical to other activators of the PRL
receptor system.
PL-I is a member of a larger family of hormones/
cytokines that comprise the PRL family. PL-I is typical
of most members of the PRL family in that it is a gly-
coprotein synthesized by trophoblast cells of the devel-
oping placenta (Soares et al. 1998). PL-I, PL-II, PL-I
variant, and PRL are all capable of interacting with
PRL receptors and activating the PRL receptor signal-
ing pathway (Soares et al. 1998). Alternatively, there
is a growing number of PRL family members that do
not interact with PRL receptors (nonclassical mem-
bers). These include PRL family members identified in
the mouse: proliferin (Jackson et al. 1994), proliferin-
related protein (Jackson et al. 1994), and likely a number
of newly discovered PRL family members (H. Müller
and M.J. Soares, unpublished results; D. Linzer, per-
sonal communication); the rat: PRL-like protein A
(PLP-A) (Deb et al. 1993), PLP-B (Cohick et al. 1997),
PLP-C (Conliffe et al. 1994; G. Dai, T. Takahashi,
and M.J. Soares, unpublished results), PLP-C variant
(G. Dai, T. Takahashi, and M.J. Soares, unpublished
results), PLP-D (Iwatsuki et al. 1996), and decidual
PRL-related protein (Rasmussen et al. 1996); and the
cow: bovine PRL-related proteins I–VI (Schuler and
Kessler 1992). Each of these ligands has unique struc-
tural properties and expression patterns within the
uteroplacental compartment during pregnancy. Unfor-
tunately, their biological actions are largely unknown.
Classic approaches involving ligand purification, label-
ing, and characterization are tedious, time-consuming,
and sometimes fraught with technical problems. On
the basis of evidence provided in this report, the gen-
eration of AP–ligand fusion proteins may provide a
relatively straightforward strategy for identifying bio-
PL-I Target Cells
743
logical targets for nonclassical members of the PRL
family.
Acknowledgments
Supported by grants from the National Institute of Child
Health and Human Development (HD 20676, HD 29797,
HD 33994). HM was supported by a fellowship from the
Deutsche Forschungsgemeinschaft of Germany (Mu 1183/1-1).
We thank Donna Millard, Bing Liu, and Belinda Chap-
man for advice and assistance in the preparation of the tissue
sections used in this study and Christopher Cohick for assis-
tance in preparing the figures. We also would like to thank
Youngsoo Lee and Dr James Voogt for providing the rat
brain tissue sections.
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