The Short Form of The Prolactin (PRL) Receptor Silences PRL Induction of the ß-Casein Gene Promoter

Juan José Berlanga1, Josefa P. Garcia-Ruiz, Martine Perrot-Applanat, Paul A. Kelly and Marc Edery

Institut National de la Santé et de la Recherche Médicale, Unité 344 Endocrinologie Moléculaire (J.J.B; M.P-A; P.A.K; M.E.), Faculté de Médecine Necker, 75730 Paris Cedex 15, France,
Departamento de Biologia Molecular-Centro de Biologia Molecular Severo Ochoa, Universidad Autonoma de Madrid, 28049 Madrid, Spain


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The PRL receptor (PRLR) is a member of the cytokine receptor superfamily. Rats and mice express two forms of PRLR, short (SPRLR) and long (LPRLR), which differ in the length and sequence of their cytoplasmic domains. We have analyzed the ability of each form of rat PRLR to transduce lactogenic signals in a bovine mammary gland epithelial cell line. The rat PRLR forms were expressed and detected by RT-PCR, indirect immunofluorescence, and cell surface ligand binding. When the biological activity of each form of PRLR was assessed by transient transfection, we found that the long form was able to activate the ß-casein gene promoter and that the short form was inactive. Interestingly, the coexpression of both forms of PRLR resulted in a block of PRL signal to the milk protein gene promoter as a function of the concentration of the SPRLR. Similar results were obtained when LPRLR was coexpressed with totally or partially inactive tyrosine mutants of either the Nb2 form or the LPRLR form. Thus, these results suggest that the SPRLR form has at least one clear biological function, i.e. to silence lactogenic signals and to contribute to a differential and acute PRL effect in rat tissues. Furthermore, the data derived from coexpression of LPRLR and PRLR mutants confirm a crucial role of the C-terminal tyrosine residue in lactogenic signaling and the dimerization of PRLRs.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The pituitary hormone PRL has been involved in a variety of biological functions, including the synthesis of milk proteins, immune responses, development of reproductive organs, osmotic balance, and behavior. The PRL receptor (PRLR) has been characterized from different species, and it is well documented that, at least in rats and mice, it is expressed as short (SPRLR) and long (LPRLR) receptor isoforms (1). These PRLR isoforms are proteins coded by a single gene and generated by alternative splicing (2). As a result, the two forms of PRLR only differ in the amino acid composition and size of the polypeptide chain beyond the first 27 amino acids of their respective intracellular domains. The transcripts of both forms are variably expressed in a tissue-specific manner, depending on the stage of the estrous cycle, pregnancy, and lactation (3, 4).

PRLRs are members of the cytokine family of receptors (1). Ligand activation of this type of receptor triggers the activation of cytoplasmic protein tyrosine kinases (5). In the case of rat PRLRs, it has been demonstrated that protein tyrosine kinases of the Jak and Src families are associated with PRLRs and are involved in the intracellular signaling of PRL (6, 7, 8, 9, 10). Functional analysis of these receptors has shown that LPRLR, as well as a third form, the Nb2PRLR, characterized in the rat lymphoma cell line Nb2 (11), are able to transduce PRL signals to the milk protein and interferon regulatory factor-1 promoters (12, 13). However, because SPRLR was inactive in the functional studies mentioned above, its function remains unclear. A recent report showed that the SPRLR is functional, being able to stimulate NIH 3T3 cell proliferation, an action that is preceded by transient activation of mitogen-activating protein kinase, while it is unable to activate the ß-casein gene promoter (14). The SPRLR shares with the LPRLR the first 27 intracellular amino acids that contain a proline-rich region (Box 1) involved in the association with the tyrosine kinase Jak2 (15). This tyrosine kinase has been shown to associate with both forms of PRLR and to play an important role in the intracellular signaling of PRL (6, 7, 8). In addition, both PRLR isoforms associate with the tyrosine kinase p59fyn (9) or the cytosolic protein tyrosine phosphatase PTP1D in Nb2 cells (16).

In this study, we assessed the biological function of the two rat PRLR isoforms by expressing them in a bovine mammary gland epithelial cell line (BMGE). When rat PRLRs were expressed using the recombinant vaccinia-T7 virus expression system, both forms were shown to be present as clusters in mammary epithelial cells. PRL signaling through each PRLR isoform was assayed by transient cotransfection with a vector containing ß-casein gene promoter. Our results show that the long form mediates PRL activation of a milk protein gene promoter while the short form is inactive, in agreement with earlier data (12). However, we found that cotransfection of cDNA from both PRLR isoforms blocked the signaling of LPRLR to milk protein gene promoter. Thus, our data strongly suggest that the short form of PRLR has a silencing role in PRL signaling to milk protein production. In addition, signaling of the LPRLR can be blocked when it is cotransfected with inactive PRLR tyrosine mutants. The silencing role of the SPRLR may have physiological significance because the expression of the two forms of PRLR is regulated in a tissue-specific manner.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PRLR Expression in Transfected BMGE Cells
To determine whether BMGE cells could be used to study the functional activity of rat PRLR isoforms, we first tested the expression of these proteins in BMGE cells by using a recombinant vaccinia-T7 virus expression system that allows a high level of expression. After infection with vaccinia-T7, cells were transfected with either pTM1-LPRLR or pTM1-SPRLR constructs. The receptor expressed was detected by indirect immunofluorescence using the anti-PRLR T1 antibody (17). As can be observed in Fig. 1Go, both PRLR forms were expressed in BMGE cells. The presence of immunofluorescent clusters can be attributed to the accumulation of overexpressed PRLR using the vaccinia-T7 virus system or to fixation conditions.



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Figure 1. Expression and Cellular Localization of Short and Long PRLR Forms in BMGE Transfected Cells

Cells were infected with vaccinia-T7 virus and then transfected with pTM1-LPRLR or pTM1-SPRLR constructs. At 14 h posttransfection, the cells were fixed and PRLR isoforms were detected by indirect immunofluorescence using T1 monoclonal antibody. A, Vaccinia-T7 virus infected, but untransfected, cells. B, Cells transfected with pTM1-LPRLR. C, Cells transfected with pTM1-SPRLR. D, Cells transfected with pTM1 LPRLR and pTM1 SPRLR.

 
To further confirm the presence of PRLR, Flag-tagged PRLR cDNA (18) was transfected in BMGE cells using the calcium phosphate method; cells were subsequently incubated in the presence (or not) of PRL and treated with anti-Flag antibodies, as previously described (18). In the absence of hormone, immunofluorescence for Flag-PRLR was observed on the cell surface in nonpermeabilizing conditions (Fig. 2aGo) and intracellularly (not shown); stimulation with ovine (o)PRL led to the disappearance of immunofluorescence from the plasma membrane as visualized using nonpermeabilizing conditions (not shown) and presence within the cytoplasm (bright dots), clearly observed using permeabilizing conditions (Fig. 2bGo); such localization probably corresponds to vesicles of the endocytic pathway, as previously described in detail in several other cell types (18). Control experiments in which cells were untransfected showed no background staining (Fig. 1AGo and Fig. 2cGo).



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Figure 2. Indirect Immunofluorescence Localization of Flag-tagged PRLR Expressed in BMGE Cells

BMGE cells transfected with cDNAs (5 µg) encoding a flag-tagged PRLR were deprived from serum overnight and fixed at 10 min–8 h after oPRL (400 ng/ml) addition (b, 20-min incubation) or without hormone (a, control), as described in Materials and Methods; control untransfected cells were also used (c). Fixation with 4% paraformaldehyde and permeabilization with methanol (-20 C) were used in panels b and c, while fixation without or with permeabilization was used to localize PRLR at the cell surface membrane or intracellularly, respectively, for both hormonal conditions. After fixation, cells were immunostained using a mouse monoclonal anti-flag antibody (10 µg/ml) as primary antibody and fluorescein-conjugated anti-mouse IgG as the secondary antibody. The flag-tagged PRLR shows expression at the plasma membrane (a, nonpermeabilizing fixation condition) in the absence of PRL, whereas it disappears from the plasma membrane in the presence of PRL in similar fixation conditions (not shown); pronounced cytoplasmic staining in all interphase cells is clearly seen, in endocytic vesicles (b) and Golgi region in the presence of PRL, using permeabilizing conditions. Obviously untransfected cells showed absence of labeling (c). Magnification, x400.

 
In addition, we analyzed by metabolic labeling the size of the PRLR forms expressed and examined native BMGE cells for the presence of any endogenous bovine PRLR. As shown in Fig. 3Go, control cells (lane 1) and cells transfected with pTM1 alone (lane 2) expressed no protein recognized by the anti-PRLR U5 (17). However, when cells were transfected with either pTM1-LPRLR (lane 3) or pTM1-SPRLR constructs (lane 4), proteins with the correct molecular mass (95 kDa and 42 kDa, respectively) were immunoprecipitated with U5 anti-PRLR antibody. Further efforts to detect any endogenous bovine PRLR mRNA in nontransfected BMGE cells by RT-PCR (Fig. 4Go) or by Northern blot (results not shown) were negative. Also, no detectable binding could be obtained in BMGE cells, whereas cells transfected with LPRLR or SPRLR constructs showed significant levels of receptors (Table 1Go). Thus, BMGE cells appear to be a good experimental system by which to study the functional activity of rat PRLR isoforms.



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Figure 3. Isotopic Labeling of Short and Long PRLR Forms Expressed in BMGE Transfected Cells

Cells were infected and transfected as described in Fig. 1Go with pTM1-LPRLR or pTM1-SPRLR constructs. They were labeled with [35S]methionine for 90 min and lysed and the lysates were subjected to immunoprecipitation with U5 monoclonal antibody. The immunocomplexes were analyzed on 7.5% polyacrylamide-SDS gels. Lane 1, normal BMGE cells; lane 2, cells transfected with pTM1 alone; lane 3, cells transfected with pTM1-LPRLR; lane 4, cells transfected with pTM1-SPRLR.

 


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Figure 4. RT-PCR Amplification of PRLR mRNA in BMGE Cells

A, Ethydium bromide staining of PCR products using primers 1 and 2. B, Hybridization of the gel shown in panel A after transfer using an internal probe. C, Ethydium bromide staining of PCR products using primers of ubiquitous cyclophilin. Lane 1, Control 293 cells transfected with LPRLR cDNA; lane 2, BMGE cells transfected with LPRLRcDNA; lane 3, BMGE cells transfected with SPRLR cDNA; and lane 4, nontransfected BMGE cells.

 

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Table 1. Binding Affinity and Number of PRLRs in BMGE Cells

 
PRL Signaling to the ß-Casein Gene Promoter in Transfected BMGE Cells
BMGE cells were used to assay PRL signals to milk proteins by each rat PRLR form. Because of the potential alterations produced on the cellular environment by the vaccinia virus expression system, it was advantageous to use a regular transient transfection system for this purpose. Each PRLR construct was transiently transfected with a ß-casein-luciferase construct by the calcium phosphate procedure and, after the recovery period, the cells were treated, with or without oPRL, for 24 h. The results obtained are shown in Fig. 5Go. As expected, the PRL signal to the ß-casein promoter was efficiently transduced by the LPRLR form and resulted in an 8-fold induction in luciferase activity whereas the SPRLR form had no activity.



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Figure 5. PRL Signaling to the ß-Casein Promoter in BMGE Transfected Cells

Cells were transiently cotransfected as described in Materials and Methods with the ß-galactosidase expression vector, the ß-casein-luciferase construct, and the PRLR forms expression vectors or empty expression plasmid as indicated. After a PRL stimulation (400 ng/ml) of 24 h, the cells were lysed, and the luciferase activity was measured and normalized to the ß-galactosidase activity. Results are expressed as fold of induction when compared with unstimulated cells and represent the mean ± SEM of three experiments.

 
SPRLR Blocks PRL Signals to the ß-Casein Gene Promoter
In rat and mouse tissues, PRLR isoforms are expressed in different proportions according to the hormonal environment associated with the estrous cycle, pregnancy, and lactation (3, 4). We tried to reproduce such in vivo situations by transiently transfecting BMGE cells with a mixture of LPRLR and SPRLR constructs in varying proportions (Fig. 6AGo) or with different amounts of the LPRLR construct alone as a control (Fig. 6BGo). The amount of transfected DNA was maintained at 6 µg by adding empty plasmid when necessary. As can be observed in Fig. 6BGo, the PRL signal was efficiently transduced to the ß-casein gene promoter at all assayed concentrations of LPRLR construct. Interestingly, cotransfection of the LPRLR:SPRLR constructs in a 5:1 ratio, decreased by 35% the transduction of the PRL signal to the milk protein gene promoter when compared with the control (Fig. 6AGo). This effect progressively increased as a function of the increase in the SPRLR expression. When the ratio of LPRLR to SPRLR was 1:5, PRL signaling to ß-casein gene promoter was completely abolished. Previous experiments have shown that increasing the ratio of SPRLR to LPRLR constructs in cotransfection experiments was effectively related to an increased ratio of expressed proteins (19). These results show a novel function of the SPRLR, which we propose to be the silencing of the LPRLR signaling to milk protein genes. The blocking effect of SPRLR was highly significant, even at the lowest ratio of LPRLR to SPRLR assayed (5:1), which suggests that PRL’s effect on cells can be efficiently modulated by regulating the expression of the PRLR isoforms.



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Figure 6. Effect of PRLR Short Form Coexpression with PRLR Long Form on PRL Induction of ß-Casein Gene Promoter

BMGE cells were transiently cotransfected as described in Materials and Methods with the ß-galactosidase expression vector, the ß-casein-luciferase construct, and different amounts of the PRLR isoform expression vectors. A, Cells were cotransfected with different proportions of PRLR long and short form expression vectors as indicated. B, Cells were cotransfected with different amounts of the PRLR long form expression vector and the empty expression vector (plasmid) as indicated. After a PRL stimulation (400 ng/ml) of 24 h, the cells were lysed, and the luciferase activity was measured and normalized to the ß-galactosidase activity. Results are expressed as fold of induction when compared with unstimulated cells, and they represent the mean ± SEM of three experiments.

 
Previous studies have demonstrated that the distal tyrosine of the LPRLR or Nb2PRLR is necessary for PRL activation of ß-casein gene promoter (20). The mutant forms of the long and Nb2 PRLR, in which the conserved distal tyrosines were mutated to phenylalanine, and which were shown to have partially or totally reduced functional activity, respectively, were used to test whether they could have a silencing role in PRL signal to milk protein gene transcription when LPRLR and these mutant forms were transiently cotransfected in cells. As shown in Fig. 7AGo, the mutant form of the PRLR in which Tyr-580 was mutated to phenylalanine (Y580F) when cotransfected with LPRLR decreased the transduction of PRL signal to ß-casein gene promoter. Interestingly, the blocking effect detected by Y580F expression was less pronounced than the one observed by SPRLR (Fig. 6AGo). However, when these studies were extended to the mutant form of the Nb2 PRLR in which the Tyr-382 was changed to phenylalanine (Y382F), the block in LPRLR signal to ß-casein gene promoter was of about the same magnitude as that detected for SPRLR (Fig. 7BGo). The reduced efficiency of Y580F to block PRL signal of the LPRLR can be attributed to the fact that this mutant receptor retains a reduced functional activity (30–50%) whereas SPRLR or Nb2PRLR Y382F has no functional activity (20). Thus, these data corroborate the essential role of the distal tyrosine residue located at the C terminus of the LPRLR for efficient PRL activation of ß-casein gene transcription and confirm a silencing role of the SPRLR.



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Figure 7. Effect of PRLR Distal Tyrosine Mutants Coexpression with PRLR Long Form on PRL Induction of ß-Casein Gene Promoter

BMGE cells were transiently cotransfected as described in Materials and Methods with ß-galactosidase expression vector, the ß-casein-luciferase construct, and different amounts of PRLR long form and PRLR mutants. A, Cells were cotransfected with different proportions of PRLR long form (WT) and Y580F mutant expression vectors as indicated. B, Cells were cotransfected with different proportions of PRLR long form (WT) and Y382F mutant expression vectors as indicated. After a PRL stimulation (400 ng/ml) of 24 h, the cells were lysed, and luciferase activity was measured and normalized to the ß-galactosidase activity. Results are expressed as fold of induction when compared with unstimulated cells, and they represent the mean ± SEM of three experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In the present study we have established a system with which to study a transcriptional signaling pathway for PRL in a more relevant cell type than the one previously used, i.e a bovine mammary epithelial cell line lacking endogenous PRLR. Our data demonstrate that in these mammary gland-derived cells, the SPRLR has at least one biological function in PRL signaling to milk protein synthesis: to silence the PRL effect, supporting a novel view in the understanding of PRL actions. These data are consistent with the evidence showing that PRL or GH activates their receptors producing a 1:2 hormone-receptor complex by a sequential dimerization (21, 22, 23, 24) and show that the composition of PRL-PRLR complexes must be important for the effect of PRL in the cells. Our results show that the PRL signal to the ß-casein gene promoter is only mediated by the formation of PRL-(LPRLR)2 complexes. This fact correlates well with previously published evidence on PRL signaling in other cell types (12, 13, 14, 15). The self-association of the intracellular domain of the LPRLR forms allows proper docking of intracellular transducer molecules at the proline-rich region (Box 1) and at the distal tyrosine of LPRLR (15, 19). This distal tyrosine is essential for PRL signaling to the milk protein genes because a LPRLR mutant for this residue results in a decrease of signal transduction (19). This could imply that either the specific amino acid sequence or the molecular space present between Box 1 and the distal tyrosine is necessary to anchor the transducer molecule that binds to distal tyrosine, since Y580F expression did not completely silence PRL signals as was true for SPRLR or Y382F. Alternatively, the quaternary structure of the PRL-PRLR complex or the associated transducer molecule may represent the structural basis for the above mentioned differences. The transducer molecule that associates with the distal tyrosine is unknown, but it has been suggested that this phosphorylated tyrosine could be involved in association with and activation of STAT5 (15, 19). When SPRLR was coexpressed with LPRLR, PRL signals to the ß-casein gene promoter were blocked in a SPRLR concentration-dependent manner. This fact strongly suggests that PRL-(LPRLR-SPRLR) complexes are inactive, which provides a rapid means of blocking PRL signals. In molecular terms, there are two possible ways to explain the inability of PRL-(LPRLR-SPRLR) complexes to transmit the PRL signal to the milk protein gene promoters. The first is that the essential transducer (STAT5) needs the two distal tyrosines provided by the self-association of the LPRLR dimers to bind the complex, while the hybrid receptor dimers have only one. The other is that this transducer could recognize only one distal tyrosine that is externally exposed in PRL-(LPRLR)2 complexes and intra- or intermolecularly hidden in the PRL-(LPRLR-SPRLR) complexes. The expression of SPRLR forms is therefore not meaningless. On the contrary, SPRLR expression provides to cells an alternate tool to modulate PRL action. Similarly, formation of homo- or heterodimers has been shown to modulate cell signaling for TNF and Fas/APO1 (25). It remains to determine whether PRL-(LPRLR-SPRLR) or PRL-(SPRLR)2 complexes are involved in other functions of PRL. Using granulocyte macrophage colony stimulating factor (GM-CSF) receptor/PRLR chimera, it has been shown recently that Nb2PRLR-SPRLR heterodimers failed to induce proliferation in BA/F3 line, and this was related to an absence of activation of Jak2 and Fyn whereas the GM-CSF/Nb2PRLR homodimers are fully capable of GM-CSF-induced proliferation and activation of Jak2 and Fyn in the BA/F3 transfectants (26). Taken together, these data indicate that the SPRLR acts, through heterodimerization, as a dominant negative isoform inhibiting both proliferation and transmission of a lactogenic signal by the receptor complex. Such heterodimers have also been shown to occur in human embryonic kidney fibroblast 293 cells after cotransfection of SPRLR and LPRLR and result similarly in an absence of activation of Jak2 (19).

New insights into the understanding of PRL effects can be derived from two facts. The first is that PRLR isoforms are expressed in a tissue-specific manner regulated by the hormonal situation associated with the stages of the estrous cycle, pregnancy, and lactation (3, 4). Second, the present data show that effects of PRL mediated by the intracellular cascade used to express the milk proteins depend on the composition of the PRLR dimers, and this concept could be extended to other functions of PRL (26) and perhaps to other growth factor receptors presenting isoforms with truncated intracellular domains (27). In the mammary gland, the LPRLR is highly expressed both at the end of pregnancy and during lactation. However, the ratio between SPRLR and LPRLR increases at midlactation with respect to the end of pregnancy. Thus, the formation of inactive PRLR heterodimers will increase at midlactation, and the PRL effect mediated by the intracellular signaling to ß-casein will decline. Thus, the SPRLR form appears to play an active negative role in PRL signaling to milk protein gene transcription.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Construction
Plasmid pTM1 was obtained from B. Moss (NIH, Bethesda, MD) (28). The DNA sequence corresponding to the LPRLR and SPRLR were amplified by PCR using the cDNAs cloned into the plasmid Bluescript SK (29, 30) as a template, after 10 cycles of amplification. The 5'-oligonucleotide primer for both cDNAs was 5'-CCGGCCGGCCATGGCATCTGCACTTGCTTT-3', and the downstream 3'-oligonucleotide primers were 5'-GGCCCCCCGGGCTATCAGTGAAAGGAGTGC-3' for LPRLR and 5'-GGCCCCCCGGGTTCAGTAGTCAAGTTCCCC-3' for SPRLR. The PCR oligonucleotides generated an NcoI site in the first AUG and a SmaI site in the 3'-untranslated sequence. The resulting PCR products were digested with NcoI and SmaI and cloned into the polylinker region of the expression vector pTM1 linearized with NcoI and SmaI.

Cell Culture and Transfection
BMGE cells (31) were grown in DMEM with 20% (vol/vol) FCS. For transfection, cells were grown in dishes of M24 plates until they reached 50–60% of confluence. They were first infected with a recombinant vaccinia virus encoding the T7 RNA polymerase (vT7) (32), at a multiplicity of infection of 5–10 in 0.2 ml serum-free DMEM for 60 min at 37 C. The inoculum was then removed, and the cells were washed twice with serum-free DMEM. Plasmids (0.5 µg/dish) were transfected by a liposome-mediated method (33) using lipofectin (GIBCO BRL, Gaithersburg, MD). Transfected cells were incubated for 14–16 h in serum-free DMEM before isotopic labeling and immunoprecipitation or indirect immunofluorescence. For Scatchard analysis, functional studies, and immunofluorescence studies with the anti-flag antibody, cells were transfected by a calcium phosphate procedure (30).

Isotopic Labeling and Immunoprecipitation
Fourteen to 16 h after DNA transfection, BMGE cells were washed twice with methionine-deficient DMEM and incubated in the same medium containing 5 µCi L-[35S]methionine (Amersham, Little Chalfont, U.K.) for 90 min at 37 C. Cells were washed twice with PBS and lysed in 0.4 ml lysis buffer [10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonylfluoride, 1% (vol/vol) Triton X-100, 2 µg/ml leupeptin, 5 µg/ml aprotinin, 1 µg/ml pepstatin A] for 30 min at 4 C. After centrifugation (15,000 x g, for 15 min at 4 C) to remove any insoluble material, the lysates were incubated with 2 µg U5 antibody (anti-PRLR monoclonal antibody) (17), 2.5 µg rabbit anti-mouse Ig (RAM, Nordic Immunological Laboratoties, Tilburg, The Netherlands), and 30 µl protein A-Sepharose (Sigma, St. Louis, MO) for 4 h at 4 C. The immunocomplexes were washed three times with lysis buffer and analyzed on 7.5% polyacrylamide-SDS gels. Gels were dried and exposed to autoradiographic films (Eastman Kodak, Rochester, NY).

Whole Cell Binding and Scatchard Analysis
Transfected cells were deprived of serum for an overnight period. For Scatchard analysis, BMGE cells transfected with PRLR cDNAs (4 µg/100-mm culture dish) were incubated with 100,000 cpm of [125I]human (h)GH and increasing concentrations (0–50 pM) of unlabeled oPRL in 1 ml PBS containing 0.5% BSA. Incubations were carried out at room temperature for 4 h. Cells were washed twice with ice-cold PBS, solubilized in 1 ml 0.5 N, NaOH and counted in a counter. [125I]hGH was prepared using chloramine T; specific activity ranged from 80–140 µCi/µg.

RT-PCR and Southern Blot Analysis
cDNA synthesis and PCR amplifications were carried out as described previously (34). For PCR amplification, we used a 25 mer (primer 1) corresponding to nucleotides 77–101 in the extracellular domain and an antisense 23 mer (primer 2) corresponding to nucleotides 796–818 in the membrane-proximal intracellular domain. Both primers are common to the long (rat, cow) and short (rat) forms of the PRLR. The amplification products were separated on 2% agarose gels, stained with ethydium bromide, and transferred onto a Hybond N+ membrane (Amersham). Southern hybridization with 5'-end-labeled oligonucleotide probe was performed according to manufacturer’s instructions. To control the efficiency of the reverse transcription, a control amplification of the ubiquitous cyclophilin was performed on the same sample as previously described (34).

Indirect Immunofluorescence
BMGE cells were grown onto coverslips and transfected as described above. Fourteen to 16 h after transfection, they were washed twice with PBS and fixed in cold methanol for 10 min at 4 C. After extensive washing with PBS, coverslips were incubated for 2 h at room temperature in blocking buffer [PBS containing 3% (wt/vol) BSA, 0.4% (vol/vol) Triton X-100], then incubated with anti-PRLR T1 monoclonal antibody (1:20 dilution) in PBS/3% (wt/vol) BSA for 2 h at room temperature. The cells were washed six times with PBS before incubation for 1 h with rabbit anti-mouse fluorescein conjugate (1:20 dilution) (17). Cells were observed with a Zeiss fluorescence microscope (Carl Zeiss, Thornwood, NY), and photographs were taken using Kodak 400 ASA film.

When using the anti-flag antibody, transfected cells were grown as subconfluent monolayer cultures in Labtek chambers. For the experiments, cells were harvested for 3–12 h in GC3 medium and incubated with oPRL (400 ng/ml) for 10 min to 12 h; cells were washed three times with PBS and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 20 min or methanol (-20 C) for 5 min. Samples were successively incubated with blocking solution, mouse anti-flag monoclonal antibody (IBI Kodak; 10 µg IgG/ml) overnight at 4 C, then washed and further incubated for 45 min with a 1:40 dilution of fluorescein isothiocyanate-conjugated goat anti-IgG. Cells were then mounted in 50% glycerol in PBS and observed on a Zeiss microscope.

Specificity control experiments included incubation of cells in the absence of primary and/or secondary antibodies or with IgG contol monoclonal antibodies. No immunofluorescence was detected in any of the specificity control experiments.

Transient Transfection and Stimulation of the ß-Casein Gene Promoter
BMGE cells were grown in a 60-mm culture dish in DMEM/20% (vol/vol) FCS until they reached 50% confluence. They were starved overnight in GC3 medium composed of 1:1 DMEM and Ham’s F-12 medium supplemented with transferrin (10 µg/ml), insulin (80 mU/ml), glutamine (2.5 mM), and nonessential amino acids. Cells were transfected by a calcium phosphate precipitation procedure (30) with 3 µg pCH110 (ß-galactosidase expression vector from Pharmacia, Uppsala, Sweden) and 1.5 µg plasmid ß-casein-luciferase (carrying a fusion between the promoter region of the rat ß-casein gene and the coding region of the luciferase gene) (20) and different amounts of expression vectors for the PRLR forms: pER11 (pECE-PRLR short form), pER23 (pECE-PRLR long form) (29, 30), flag-tagged PRLR (19), pCMV-WT (pCMV-PRLR long form), pCMV-Y580F (pCMV-PRLR long form mutant Y580F), and pCMV-Y382F (pCMV-PRLR Nb2 form mutant Y382F) (20). After a glycerol shock, fresh GC3 medium was added and the cells were incubated in the presence or absence of 18 nM oPRL (400 ng/ml) and dexamethasone (250 nM) for 24 h before being lysed. Luciferase activity was measured in arbitrary light units and normalized to the ß-galactosidase activity. Results of luciferase activity of ß-casein promoter upon PRL stimulation (18 nM) are expressed as fold induction compared with cells not stimulated with PRL. Results represent the mean ± SEM of three different experiments.


    ACKNOWLEDGMENTS
 
We especially thank Oreste Gualillo for his help with the transfections, Josefa González-Nicolás for her assistance with the photography, and Dr. E. Martinez-Salas for a critical review of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. Marc Edery, Institut National de la Santé et de la Recherche Médicale, Unité 344 Endocrinologie Moléculaire, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris, Cedex 15 France.

This work was supported by grants from Dirrección General de Investigacion Científica y Técnica (PB93–0255) and the Ramon Areces Foundation. The Universidad Autonoma de Madrid supplied financial support to J.J. Berlanga for a short-term visit to the Unité 344 of INSERM, Paris, France.

1 Current address: Departamento de Biologia Molecular-Centro de Biologia Molecular Severo Ochoa, Universidad Autonoma de Madrid, 28049 Madrid, Spain. Back

Received for publication November 15, 1996. Revision received June 9, 1997. Accepted for publication June 17, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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