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Department of Comparative Biosciences, University of Wisconsin (L.A.S., R.N., M.A.K.), Madison, Wisconsin 53706; and the Department of Molecular and Cellular Physiology, University of Cincinnati (J.G., N.D.H.), Cincinnati, Ohio 45267
Address all correspondence and requests for reprints to: Dr. Linda A. Schuler, Department of Comparative Biosciences, 2015 Linden Drive West, University of Wisconsin, Madison, Wisconsin 53706. E-mail: schulerl{at}svm.vetmed.wisc.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is now clear that the PRLR is not a single uniform entity. A so-called long form of the PRLR (lPRLR), which has been found to readily transmit signal in all systems studied, has been characterized in multiple species. Like other members of the cytokine receptor superfamily, PRLR has no intrinsic kinase activity, but has been shown to associate with other molecules (for reviews, see Refs. 6 and 7), which may vary with cell type and the nature of the signal. PRL-stimulated ß-casein gene transcription, which is mediated by JAK2 and activation of the transcription factor STAT5 (signal transducer and activator of transcription-5) (8, 9), is perhaps the most studied model system. The rat PRLR requires the most carboxy tyrosine residue (Tyr580) to transmit this signal (10). However, this may not be true in all species. The coding sequences for the lPRLR in the cow (4), sheep (11), and red deer (12) contain an in-frame termination signal before the codon for this tyrosine, which results in a protein about 40 amino acids shorter than that reported for the human, rabbit, and rodent receptors (13, 14, 15).
In addition to the lPRLR, short forms of the PRL receptor (sPRLR) with truncated cytoplasmic domains have been characterized in rats and mice (14, 15, 16). The existence of this alternative form outside the rodent species was questioned for some time; however, recently, a sPRLR was also reported in the sheep (5, 11). The function of the sPRLR is not yet understood; it has been shown to be inactive in transcriptional activation of both the ß-casein (17, 18) and interferon regulatory factor-1 (19) promoters. However, the sPRLR has been shown to mediate proliferation in NIH 3T3 cells transfected with this receptor (18), although not in FDC-P1 cells (19). In Ba/F3 cells transfected with chimeric receptors, the short form was able to competitively inhibit lPRLR chimera-mediated proliferation, as well as JAK2 and Fyn activation (20). Gibori and her colleagues have identified an ovarian phosphorylated protein associated with the sPRLR (21), suggesting that in these cells, at least, the sPRLR may activate a distinct signal transduction pathway.
The ligands presented to maternal and fetal PRLR during pregnancy are also heterogeneous. In the fetus, the PRLR ligands include PRL secreted as the fetal lactotrophs mature during the latter part of gestation as well as species-specific placental lactogens (PLs) secreted by the trophoblast (for reviews, see Refs. 22 and 23). Ligands available to maternal PRL receptors include pituitary PRL, decidual PRL, and related placental and decidual hormones, which also vary with species. Temporal patterns of expression of the placental and decidual hormones and their selective secretion into the different compartments as well as progressive maturation of fetal lactotrophs during gestation result in changing absolute and relative concentrations of these ligands during pregnancy. Accumulating evidence indicates that rodent PLs are good agonists at the PRLR (24, 25); this is less clear for the related placental hormones in other species. In ruminants, bovine PL (bPL) is present in the fetal circulation at peak concentrations during midgestation (for a review, see 26 before the fetal lactotrophs are fully mature. The ability of bPL to activate the bovine PRLR has not been resolved; however, our laboratory has shown that bPL can bind the bovine PRLR with equal or greater affinity than PRL itself (4).
To increase our understanding of the actions of PRL and related hormones during pregnancy across species, we examined various putative target tissues in both fetus and mother for receptor protein and messenger RNA (mRNA) and determined the effect of glycosylation on the apparent sizes of these proteins. To investigate the ability of the PRLR forms and their ligands to mediate signals leading to transcriptional activation, we employed a PRL response element reporter gene system to examine the ability of both the long and short forms of the receptor to mediate PRL and bPL signaling. These findings will facilitate study of the tissue-specific actions of PRL and related hormones during pregnancy.
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Materials and Methods |
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Identification of the alternative short PRLR form
To ascertain whether cows, like mice (14), rats (16), and sheep
(5), express a form of the PRLR with a truncated cytoplasmic domain,
primers were designed from the bovine PRLR sequence (4) to span the
location of the insertion leading to the alternate form in sheep. The
upstream primer (5'-CCAGAGAGCTCCATCCAGATACCTA-3') is located proximal
to the bases encoding the transmembrane domain (beginning at nucleotide
743 of the published sequence), and the downstream primer
(5'-CAAGTCCTCGCAGTCAGAAGTGG-3') is located 3' to the site of insertion
(beginning at nucleotide 1016) in the sheep mRNA. All primers were
obtained from the University of Wisconsin Biotechnology Center. Reverse
transcription-PCR, as previously described (4), was used to amplify
this region of the PRLR transcript from pregnant endometrium. This
procedure yielded the predicted product of 274 bp resulting from
amplification of the long form of the receptor as well as a larger
product of 313 bp. The distance between these primers was similarly
examined in previously isolated complementary DNA (cDNA) clones
prepared from a pregnant endometrial library, and the region of
interest was sequenced as previously described (4).
Determination of the relative amounts of the short and long forms
of the receptor
cDNA was prepared from various tissues after priming either with
oligo(deoxythymidine) for those tissues with relatively abundant PRLR
mRNA (maternal liver, endometrium, and corpus luteum) or with the
12-mer (5'-CCAGAGTCACTG-3'), which is complementary to both forms of
the bPRLR, for those tissues in which PRLR transcripts are less
abundant. The latter strategy has been successfully employed to
increase cDNA for low abundance transcripts such as the PRLR (27).
Reverse transcriptase products derived from 750 ng total RNA were
amplified by PCR with the primers that flank the insertion site
described above for 35 cycles. Analysis of the kinetics of product
formation confirmed that both short and long PRLR cDNAs were amplified
at approximately the same rate, and that product formation was
exponential through at least 40 cycles under these conditions. PCR
products were separated with 3% NuSieve (FMC BioProducts, Rockland,
ME)-1% agarose gels, transferred to nylon filters, and hybridized to
32P-labeled PRLR cDNA. Signals were quantitated with the
Ambis Radioanalytic Imaging System (San Diego, CA). Three samples of
each tissue type were examined, and each determination was repeated
three times.
Production of a polyclonal antibody to the bPRLR
The extracellular domain of the bovine PRL receptor (4) was
expressed as a fusion protein from a modified pGEMEX-I vector (Promega,
Madison, WI). The vector was modified by insertion of a double stranded
factor Xa protease cleavage site oligonucleotide into the
KpnI/SacI site of pGEMEX-I. The resultant
pGEMEX-Xa vector was further digested with ClaI, Klenow
treated, and religated. This construct was then digested with
EcoRV/SacI and ligated with the region encoding
amino acids -1 through 272 (the entire extracellular domain,
transmembrane domain, and proximal cytoplasmic domain until
approximately after box 1) of the bPRLR (4) to yield expression
construct 367. In this construct, the T7 promoter drives transcription
of RNA coding for T7 gene 10, translationally fused to a downstream
factor Xa cleavage site preceding the bPRLR sequence.
The 367 expression plasmid was transfected into Escherichia coli strain JM109 DE3. Five-milliliter cultures of the plasmid-bearing strain were grown overnight at 37 C in M9 medium supplemented with 0.5% glucose and 2% casamino acids. The following day, the overnight cultures were inoculated into 1-liter flasks of Luria-Bertoni medium supplemented with 50 µg/ml ampicillin. When the A600 of the culture reached 0.5, isopropyl ß-D-thiogalactopyranoside was added to a final concentration of 1 mM. After 4 h, the cells were pelleted and frozen at -80 C until use. The fusion protein in inclusion bodies was isolated according to the method of Nagai and colleagues (28). The insoluble PRLR fusion protein was solubilized in 8 M urea, 10 mM Tris-Cl (pH 8.0), and 1 mM EDTA and loaded on a S-300 column equilibrated with 6 M urea, 1 mM EDTA, 15 mM Tris-Cl (pH 8.8), 100 µM ß-mercaptoethanol, 0.02% Triton X-100, and 0.02% NaN3. The peak fractions were analyzed by Coomassie-stained SDS-PAGE gels, pooled, and dialyzed against 20 mM Tris-Cl (pH 8.0), 100 mM NaCl, and 2 mM CaCl2. The fusion protein was concentrated and digested overnight with factor Xa (New England Biolabs, Beverly, MA) at room temperature. The products were separated on a 15% SDS-PAGE gel and stained with 0.25 M KCl. The band representing the extracellular domain of the PRLR (confirmed by amino acid sequencing, University of Wisconsin Biotechnology Center) was excised, and two rabbits were immunized with 250 µg protein. Antibody titers were monitored by Western analysis.
The antibody was purified by affinity chromatography. The 367 fusion protein and the gene 10 protein were coupled to separate columns (Reacti-gel 6X, Pierce Chemical Co., Rockford, IL) according to the manufacturer’s instructions. The immune rabbit serum was diluted 10-fold with PBS and passed over the gene 10 column three times. The flow-through was collected and then passed over the 367 fusion protein column three times. The 367 column was washed sequentially with low salt (10 mM Tris-Cl, pH 7.5) and high salt (10 mM Tris-Cl, pH 7.5, and 500 mM NaCl) buffers. Bound protein was eluted with 100 mM glycine, pH 2.5. The fractions were immediately neutralized with 2 M Tris-Cl, pH 8.0, and concentrated in a Centriprep 10 (Amicon, Beverly, MA).
To confirm that our antibody recognized the appropriate antigen, the long form of the PRLR (4) was transcribed and translated in vitro using the Novagen (Madison, WI) Single Tube Protein System according to the manufacturer’s instructions. The product was immunoprecipitated using preimmune or purified antibody and protein A-Sepharose, fractionated by SDS-PAGE, and visualized by autoradiography. In addition, antibodies selected with the preparation containing the sequenced PRLR antigen detected proteins of approximately the expected sizes in both transfected cells and microsomal membrane preparations, whereas selection with preparations containing the G10 protein alone, other unrelated antigens (GH receptor and ATP2), or preimmune serum did not.
Western analysis of solubilized tissue microsomes
Tissue microsomes were prepared as previously described (29).
Aliquots of the microsomes were solubilized in 25 mM HEPES
(pH 7.4), 0.25% Nonidet P-40, 2 mM
Na3VO4, 10 µg/ml aprotinin, and 1
mM phenylmethylsulfonylfluoride and centrifuged at
100,000 x g for 1 h at 4 C. The supernatant was
aliquoted and frozen at -80 C until use. Ninety micrograms of
microsomal protein were fractionated by 7.5% SDS-PAGE (30). Proteins
were electrophoretically transferred to polyvinylidene difluoride
membranes, blocked in TBST [10 mM Tris-Cl (pH 7.4), 135
mM NaCl, and 0.1% Tween-20] containing 0.25% gelatin for
at least 2 h, and then incubated in either the PRLR antibody
(1:1,000 dilution of original sera) or a control antibody. After a
brief wash in TBST, the membranes were incubated in antirabbit IgG
conjugated to horseradish peroxidase (1:10,000) for 1 h. Proteins
were visualized by enhanced chemiluminescence (Amersham Life Sciences,
Arlington Heights, IL).
Transient transfection of COS-7 and END-6.2 cells
Both forms of the bPRLR were cloned into the expression vector,
pcDNA3 (Invitrogen), and transfected into COS-7 or END-6.2 cells by
electroporation as previously described (4). The END-6.2 cells are a
bovine endometrial stromal cell line derived from conditional
transformation of pregnant endometrial cells with a
temperature-sensitive simian virus 40 T antigen construct (31, 32).
Twenty-four hours after transfection, cells were treated with buffer or
tunicamycin (2 µg/ml). After an additional 48 h, cells were
harvested. Ninety micrograms of cell protein were fractionated by
SDS-PAGE, and immunoreactive bPRLR were examined as described
above.
Stimulation of transcription by the PRLR forms
The reporter construct PRE3-chloramphenicol transferase
(PRE3-CAT) (33), which contains three copies of the PRL response
element from the rat ß-casein gene upstream from the CAT gene under
the control of the minimal promoter of the herpes simplex virus
thymidine kinase gene, was used to examine the ability of these PRLR
forms and the physiological ligands, PRL and bPL, to mediate a
transcriptional response. CHO-K1 or COS-7 cells were maintained in
Ham’s F-12 or DMEM medium, respectively, containing 10% FBS.
Twenty-four hours before transfection, cells were seeded at a density
of 106 cells/100-mm dish. Cells were cotransfected by
calcium phosphate precipitation, as previously described (33), with 5
µg pigeon or bovine PRLR expression constructs, 5 µg reporter
construct, 5 µg ß-galactosidase expression vector (pRSVZ) to
monitor transfection efficiency, and 10 µg salmon sperm DNA. Four
hours later, cells were shocked for 3 min in DMEM containing 10% FBS
and 10% glycerol and then fed serum-free medium containing hormone (4
nM) or buffer control. Forty-eight hours later, cells were
harvested in 0.25 M Tris-HCl, pH 7.8, lysed by four
freeze-thaw cycles, and centrifuged at 12,000 x g for
10 min. Supernatants were assayed for ß-galactosidase and CAT by
enzyme-linked immunosorbent assay (Life Technologies, Gaithersburg, MD)
according to the manufacturer’s instructions. All data were normalized
for transfection efficiency using ß-galactosidase activity and
presented as the fold induction over the untreated control values.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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50 amino
acids compared with 360 amino acids for the long forms) were among
the first PRLR to be cloned. In contrast to this apparent two-receptor
system in rodents, studies in other species had identified only a long
form of the PRLR. However, recently, Anthony (5) and Bignon (11) and
their colleagues described a short form of the receptor in sheep,
consistent with a broader role for the sPRLR in mammals other than
rodents (diagrammed in Fig. 1
).
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). This transcript predicts a PRLR of 272 amino acids,
with a mass of 33,840 Da. The entire transmembrane and proximal
cytoplasmic region in both forms of this receptor are very similar to
those in the sheep PRLR (95% nucleotide identity, nearly identical
amino acid sequence except for 2 conservative amino acid
substitutions).
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and 2, the abbreviated bovine receptor diverges
from the long form at precisely the same location as the rat (16), all
three murine forms (15), and the ovine short form (5). They all include
the proline-rich region (box 1), which was shown to be necessary for
JAK2 activation (34) but not sufficient for transcriptional activation,
but not the next highly conserved region (box 2). Note that the bovine,
ovine, and one murine (PR-1), but not the other murine, sPRLR (PR-2 and
-3) have only a single Tyr residue in their cytoplasmic domains, in
contrast to the other murine sPRLR forms and the single reported rat
form, which contain three, three, and four, respectively. Transient transfection of the bovine sPRLR into COS-7 cells demonstrated that it was similar to the long form with respect to affinity for both bPRL and bPL (Kd, 2.0 x 10-10 M).
Reverse transcription-PCR was employed to determine the relative
abundance of these forms of PRLR transcripts in various fetal and
maternal tissues. As shown in Fig. 3B, all tissues
examined contained both kinds of transcripts, but the ratio varied
among tissues. With the exception of maternal liver, corpus luteum,
and, to a lesser extent, maternal intestine, the mRNA encoding the long
form of the receptor was the most plentiful in all tissues. Unlike the
adult liver, the fetal liver notably expresses primarily the long form
of the receptor.
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, this affinity-purified antibody specifically
recognizes PRLR transcribed and translated in vitro (Fig. 4A) as well as both forms of the receptor transiently transfected into
COS-7 cells (Fig. 4, B and C).
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, immunoreactive PRLR shows considerable heterogeneity in apparent size
and relative abundance in the different tissues examined. All samples
revealed one or multiple immunoreactive protein moieties with apparent
mol wt of about 100,000 or greater, which in some tissues (fetal liver,
spleen, and thymus; maternal endometrium and peripheral blood
lymphocytes) comprised the major signal(s). Maternal endometrium
demonstrated an apparently single immunoreactive species of about
100,000; however, fetal liver, spleen, and thymus as well as maternal
intestine and liver demonstrated a band of approximately 110,000, and
fetal brain, liver, and thymus and maternal liver exhibited a slightly
larger band of about 120,000. Fetal thymus showed an additional larger
band, about 130,000, which was also found in maternal peripheral blood
lymphocytes, where it was the major immunoreactive species. Smaller major immunoreactive signals were observed at a apparent mol wt of about 72,000 in the fetal brain; about 55,000 in fetal thymus, maternal intestine, corpus luteum, and endometrium; about 50,000 in maternal liver; and about 47,000 in fetal liver. Other minor bands in these size ranges were present in the various microsomal samples.
Comparison of the relative transcript levels to the Western analysis reveals a good correlation between the relative amounts of PRLR proteins detected and mRNA present, if the immunoreactive species of 100,000 mol wt or larger are presumed to be modified long forms of the receptor, and those of 55,000 mol wt and smaller are presumed to be modified short forms.
To examine the contribution of N-linked glycosylation to the
mobility of the receptor proteins, cell lines transiently transfected
with one or the other receptor form were cultured with and without
tunicamycin. As shown in Fig. 4B, Western analysis of COS-7 cells
transfected with the short form of the receptor revealed two
immunoreactive bands with apparent mol wt of about 41,000 and 46,000.
Inhibition of N-glycosylation decreased the mobility of each
band by about 10,000, the smaller of which was similar to the predicted
mass of the short form (33,800 Da). The lPRLR transfected into these
same cells cultured without tunicamycin was detected as two forms of
about 88,000 and 102,000 (Fig. 4C). Tunicamycin treatment reduced them
to major species of 81,000 and 95,000 (predicted mass from cDNA,
63,000 Da). In contrast, the bovine stromal endometrial cell line,
END-6.2, cultured without tunicamycin displayed a doublet of about
90,000 and 97,000, which reduced to a single band of about 83,000 in
the presence of tunicamycin (Fig. 4D, lanes 1 and 2). The larger of
these bands was similar in mobility to that detected in endometrial
microsomes, which represent a more heterogeneous cell population (Fig. 4D, lane 3). These data suggest that N-linked carbohydrate
contributes to the apparent size of the receptor; however, the
inability of tunicamycin to restore the mobility of the lPRLR and a
portion of the sPRLR signals to that predicted from the cDNA and
comparison of the different mobilities in COS-7 and END-6.2 cells as
well as tissue microsomes indicate that it is not the only
modification.
Finally, we examined the ability of these receptor forms to stimulate
transcription in an established reporter gene assay (33). As shown in
Fig. 5, the long form of the bPRLR mediated stimulation
of the PRE3-reporter gene construct in CHO-K1 cells in response to both
bPRL and bPL. There was no significant difference in the response to
these ligands. Both the bovine lPRLR and pigeon PRLR mediated PRL
stimulation of the 2.8-kilobase ß-casein promoter in CHO-K1 cells
with a similar fold increase over that in control cultures, although
the basal and stimulated levels were about 2-fold lower than with the
PRE3-reporter gene construct (data not shown). Similar assays of
transfected COS-7 cells demonstrated that the ability to stimulate
transcription in this assay depended on STAT5 (data not shown), as
reported for the pigeon PRLR (33). In contrast, the short form of the
bPRLR was unable to stimulate transcription despite similar specific
binding of ligand to these cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PRLRs have been examined by cross-linking and/or Western analyses from
multiple tissues, including liver, mammary gland, ovary, gastric
mucosa, extraembryonic membranes from several species (35, 36, 37, 38, 39, 40), as well
as T47D (41) and insect Sf9 cells (42). In these studies, generally two
sizes of PRL-binding proteins have been detected, a larger
85,000–90,000 apparent mol wt protein and/or a smaller 40,000–42,000
moiety(s). Although our studies also revealed larger (apparent mol wt,
100,000 or greater) and smaller immunoreactive molecules
(47,000–55,000), these sizes are somewhat larger than those observed
for other species. However, they generally correlate very well with the
mobility of the bovine PRLR expressed in COS-7 and END-6.2 cells,
supporting a correspondence to the products of the two PRLR
transcripts. In addition, we observed intermediate sized proteins,
particularly in the fetal brain, which, although still larger than the
predicted size of the unmodified lPRLR, were considerably smaller than
the larger immunoreactive moieties in both tissue microsomes and
transfected cells. Multiple proteins of other sizes have also been
reported in some studies (38, 39, 40). It is unclear if these represent
proteins modified very differently posttranslationally (e.g.
proteolysis), or products of yet another PRLR transcript such as that
observed in human mammary tissue (43), or proteolytic artifacts despite
rapid tissue collection and use of protease inhibitors.
Perhaps some of the differences in apparent size between the bovine PRLR and receptors in other species are due to differences in glycosylation. Because the sPRLR is the most abundant in the adult liver, which has the highest level of PRLR, it has been the most extensively studied. The rat sPRLR has been found to have only N-linked and no O-linked carbohydrate groups (35). In this species, all three consensus sites for glycosylation appear to be used, and together contribute about 8,000–10,000 to the apparent mobility on SDS-PAGE (35, 44). Structures of the attached carbohydrate moieties have been shown to differ between the mouse and rat liver (36). The bPRLR encodes only two consensus sites for N-glycosylation in its extracellular domain, although the bovine sPRLR transfected into COS-7 cells shows a similar total contribution of carbohydrate to its mobility as other species. This suggests a different carbohydrate structure that retards mobility more than reported for other species, at least in this cell type. N-Linked glycosylation is apparently not the only modification of the bovine receptors, as even when cultured in the presence of tunicamycin, COS-7 cells transfected with both forms of the bPRLR express two apparent sizes of the proteins, differing by about 10,000 in apparent mol wt. The single immunoreactive band observed in the presence of tunicamycin in transfected END-6.2 cells suggests that cell-specific differences may also be a factor in the varying sized moieties observed in the present studies, but this requires further investigation.
Many of the immunoreactive PRLR species detected in these studies are apparently larger than those observed in the transiently transfected COS-7 cells. Whether this represents cell-specific modifications in the surface receptor or molecules linked in processing pathways has yet to be determined. Because the microsomal fraction contains organelles other than plasma membrane, it is not possible from these studies to determine which of the different mobility forms of the receptor are presented on the cell surface and which are due to processing of newly synthesized receptor or after ligand binding. Ubiquitin has been reported to be linked to the PRLR expressed in insect cells (42) and the related GH receptor isolated from rabbit liver (45). Ubiquitin conjugation was recently found to play a role in ligand-stimulated endocytosis of the latter receptor (46). It is noteworthy that in the present studies, although culture in tunicamycin resulted in a sPRLR similar in mobility to that predicted from the cDNA, indicating that N-linked glycosylation was the primary modification altering the mobility of this protein, similar treatment of both COS-7 and END-6.2 cells transfected with the lPRLR did not increase mobility to a similar extent. This suggests additional modifications on the cytoplasmic domain of the lPRLR that are not found on the truncated cytoplasmic domain of the sPRLR.
Differences in glycosylation as well as other posttranslational modifications may also alter ligand affinity, which may be particularly important in fetal tissues exposed to both PL and PRL. Although Rozakis-Adcock and Kelly (44) observed no major role of the carbohydrates in PRL binding, this does not rule out the possibility of small differences in modulating affinity. Lascols and colleagues (36), in their comparison of the carbohydrate additions to the rat and mouse liver receptors, detected differences in sialylation that correlated with small differences in ligand affinity.
Examination of the ability of these bPRLR forms to mediate signal transduction through the PRL response element via STAT5 confirmed that the bovine short form, like that of rats, is unable to stimulate transcription via this pathway. In contrast, the long form of the bPRLR was very effective in this assay despite the absence of the final Tyr residue in the bPRLR, which was shown to be essential for signal transduction in a similar system with the rat receptor (10). It is likely that the bovine receptor employs another tyrosine to bind STAT5. Edery and colleagues reported that a mutated rabbit receptor lacking this Tyr residue was able to activate transcription (47), and the PRLR of the red deer, which also lacks this final residue, can activate JAK2 and the ß-casein promoter (12). Similarly, mutagenesis of other members of the cytokine receptor superfamily, receptors for erythropoietin and GH, demonstrated that several Tyr residues were able to mediate STAT5 stimulation (48, 49). Determination of which Tyr in the bPRLR mediates this event is under study; it is noteworthy that the positions of few of the Tyr in the rat and human lPRLR are conserved in the bovine receptor.
Our studies have shown that bPL, like the PLs of rodents, is capable of activating transcription through the homologous PRLR. Gertler and his colleagues have examined the interaction of bPL with bPRLR and found much lower binding of bPL than PRL to this receptor (50), in contrast to our earlier report (4). How these studies can be reconciled is unclear. The bPRLR examined by Gertler and his colleagues differs from that used in the present studies at two amino acid positions. In addition, our respective groups have examined PRLR in different cells, and as we have observed in the studies reported here, there may be differences in the posttranslational modification of these receptors in different tissues. Finally, the preparation of ligand they used was different from ours. Any differences must be interpreted with caution because the differences in primary structure of the pituitary and placental ligands as well as their individual posttranslational modifications may result in different receptor interactions at different targets.
The diverse tissue-specific actions of PRL and related hormones, and the signaling pathways that mediate their effects are just beginning to be examined. Development of cell-specific in vitro model systems will allow dissection of its actions at fetal and maternal targets. Analysis of in vivo models such as the PRLR knock-out transgenic mouse (51) in different genetic backgrounds will facilitate appreciation of its actions in context.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Pathology, Geisinger Medical Center,
Danville, Pennsylvania 17822.
Received February 3, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T. E. Spencer, A. Gray, G. A. Johnson, K. M. Taylor, A. Gertler, E. Gootwine, T. L. Ott, and F. W. Bazer Effects of Recombinant Ovine Interferon Tau, Placental Lactogen, and Growth Hormone on the Ovine Uterus Biol Reprod, December 1, 1999; 61(6): 1409 - 1418. [Abstract] [Full Text]
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