©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Proline-rich Sequence-mediated Jak2 Association to the Prolactin Receptor Is Required but Not Sufficient for Signal Transduction (*)

Jean-Jacques Lebrun (1), Suhad Ali (2), Axel Ullrich (2), Paul A. Kelly (1)(§)

From the (1) From INSERM Unite 344, Endocrinologie Moléculaire, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France and the (2) Department of Molecular Biology, Max-Planck Institute fur Biochemie, Am Klopferspitz 18A, 8033 Martinsried, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The prolactin receptor (PRLR) belongs to the superfamily of cytokine/growth hormone/prolactin receptors. Members of this family do not contain a tyrosine kinase domain but are associated with cytoplasmic kinases of the Jak family. Here, we examine different mutants of the PRLR with respect to their ability to associate and activate the kinase Jak2 and the transcription factor Stat1. Moreover, using a biological assay system we are able to correlate these activities with activation of prolactin-responsive gene transcription. Our results indicate that interaction between Jak2 and PRLR requires a proline-rich sequence in the membrane proximal region of the receptor, which is conserved among the different members of the cytokine receptor superfamily. We also show that association of Jak2 with the receptor is sufficient for activation of the kinase as well as the transcription factor Stat1. Moreover, our findings indicate that association of PRLR with Jak2 is necessary but not sufficient for the transmission of a lactogenic signal. We have identified two other cytoplasmic regions of the PRLR that are required for activation of transcription. These two regions are located between boxes 1 and 2 and are in the carboxyl-terminal tail of the receptor. These sites probably involve specific interactions with other effector molecules.


INTRODUCTION

The prolactin receptor (PRLR)() belongs to the recently described superfamily of cytokine/GH/PRL receptors (1) , which share several structural features, including a unique transmembrane domain, an extracellular region containing four conserved cysteine residues, and a WS XWS motif. The cytoplasmic domains of these receptors, which contain no intrinsic kinase function, show very low overall sequence identity. The membrane proximal region (100 amino acids) of the receptors from this superfamily includes two conserved motifs: box 1, which contains a proline-rich domain, known to be involved in protein-protein interactions (2) , and box 2, which does not contain any known consensus sequence. It has been shown for many members of the family that mutations in this membrane proximal region result in a loss of functional activity, which suggest that it plays a crucial role in signal transduction.

Recently, efforts to identify signal transducers activated by the cytokine/GH/PRL receptor family have demonstrated that cytokine receptors associate with and activate several cytoplasmic tyrosine kinases of the Janus tyrosine kinase family (3) . It has been shown for both the GHR and the PRLR that this region including the first 85 amino acids (aa) and 94 aa of the cytoplasmic domain, respectively, was also necessary for Jak2 association with these receptors (4, 5) . In addition, recent reports also showed for the EPOR and the -chain of the GM-CSFR that the region between the two boxes was necessary for the interaction between the kinase and the receptor (6, 7) . These data indicate that the site of interaction between the tyrosine kinases from the Jak family and the cytokine receptors is located in this extended juxtamembrane region.

The cDNA encoding the PRLR has been cloned from a number of different species (8) and exists in several natural forms. In the rat, two major forms have been characterized: a short form of 291 aa with a cytoplasmic domain of 57 aa (9) and a long form of 591 aa with a cytoplasmic region of 357 aa (10) . These two forms share the same extracellular and transmembrane domains and differ only in their carboxyl-terminal cytoplasmic domain. In addition, an isoform of the rat PRLR has been identified in the rat lymphoma cell line Nb2, representing a deletion of 198 aa compared with the long form of the receptor (11) . Functional analysis of the three different natural forms of the PRLR revealed that only the long and Nb2 forms are able to induce promotor activity of the -casein promotor (12, 13) as well as the prolactin-inducible T cell activation gene, interferon regulatory factor-1 (14) . These results suggest that the cytoplasmic regions common to the long and Nb2 forms are sufficient for signal transduction.

Prolactin, similar to other cytokines, may regulate gene transcription through activation of the newly identified family of transcription factors designated as signal transducers and activators of transcription (Stat) (3) . Following their phosphorylation, these homologous and heterologous multiprotein complexes acquire the ability to translocate to the nucleus and induce transcription of ligand-responsive genes. Recently, prolactin has been shown to activate two members of this Stat family, Stat1, also known as p91, and Stat5, also called mammary gland factor (15, 16, 17) . The role of Stat5 in activation of -casein gene transcription has recently been examined (17) . However, the role of Stat1 activation in this signaling pathway remains unknown.

In this report, we have studied the molecular properties of the different forms of the PRLR as well as several deletion and truncation mutants for their ability to associate and activate the tyrosine kinase Jak2 and to undergo tyrosine phosphorylation. The capacity of these receptor constructs to induce prolactin-dependent transcriptional activation of the -casein gene promotor and to activate a transcription factor Stat1, known to be involved in PRLR signaling, was also examined. Our results indicate that the PRLR-associated kinase Jak2 binds to the proline-rich region of the receptor and that this interaction is absolutely necessary for tyrosine phosphorylation of the kinase, the receptor, and the transcription factor Stat1. However, our results also demonstrate that these activities are not sufficient for prolactin-mediated transcriptional activation of the -casein gene promotor. Finally, we have characterized two other regions of the PRLR cytoplasmic domain including the region between box 1 and box 2 and the carboxyl-terminal tail of the receptor to be necessary for activation of milk protein gene transcription.


EXPERIMENTAL PROCEDURES

Materials

Ovine prolactin (oPRL) (NIDDK) was obtained from the National Hormone and Pituitary program/NIDDK (Baltimore, MD). Recombinant bovine GH was kindly provided by Dr. W. Brumbach (American Cyanamid Co., Princeton, NJ) (ref 9450-1-7).

Cell Culture and Transfection

The 293 fibroblast cells and the stable clone expressing the tyrosine kinase Jak2 (clone LA) were grown in DMEM nut F12 medium containing 10% fetal calf serum. Several hours before transfection, cells were plated in a rich medium ( DMEM nut F12, DMEM 4.5 g/liter glucose) containing 10% fetal calf serum. Then, the cells (5 10) were transfected with the indicated amounts of cDNAs encoding Jak2 (kindly provided by Dr. J. Ihle), Stat1 (cloned from a human placenta library), and the different forms of PRLR. After 24 h of expression, the cells were deprived of serum for an overnight period.

Establishment of a 293 Cell Line Stably Expressing the Tyrosine Kinase Jak2 (Clone LA)

293 cells at 50% confluency were cotransfected with an expression vector encoding the tyrosine kinase Jak2 and an expression vector (pSVneo) encoding the resistance gene neomycine (Pharmacia Biotech Inc.) at a 10/1 ratio using the calcium phosphate technique. 2 days following transfection, medium was changed, and fresh medium containing G418 at 500 µg/ml was added; after three weeks, 70 resistant colonies were selected, and Jak2 expression in these cells was examined by Western blotting with a polyclonal anti-Jak2 antibody (UBI). We selected several clones expressing different levels of the kinase Jak2 and used the LA clone in the present study, which expresses a high level of Jak2.

Cell Surface Labeling of the PRLR

The PRLRs expressed at the cell surface were labeled by incubation with a monovalent Fab fragment generated from a monoclonal antibody (U5) directed against the extracellular domain of the PRLR at 0.5 µg/ml for 20 min at 37 °C before stimulation.

Purification of the PRLR Complexes

Cells were stimulated with either oPRL or biotinylated oPRL and recombinant bovine GH for 10 min at 5 10 M. Then, the cells were lysed in 1 ml of lysis buffer (10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 10% glycerol, 0.5% Triton X-100) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin A, 2 µg/ml leupeptin, 5 µg/ml aprotinin) for 10 min at 4 °C. Then, the insoluble material was discarded by centrifugation at 12,000 g for 5 min, and the amount of protein was equalized in all the samples by a protein concentration measurement using the Bradford technique. PRLR complexes were then incubated with 10 µl of avidin-agarose beads (10% w/v in lysis buffer) for 2 h at room temperature for the samples stimulated by biotinylated hormones or with 10 µl of anti-IgG-agarose beads (10% w/v in lysis buffer) at 4 °C for the samples labeled with the Fab monovalent fragment. Samples were washed three times in 1 ml of lysis buffer and eluted in 20 µl of SDS loading buffer (20% glycerol, 10% -mercaptoethanol, 4.6% SDS, 0.125 M Tris-HCl, pH 6.8).

Immunoprecipitation with an Anti-Stat1 Antibody

Cell lysates were immunoprecipitated for 3 h at 4 °C with 2 µl of Stat1 polyclonal antibody raised against the peptide CPEEFDEVSRIVGSVEFD and 10 µl of protein A-Sepharose beads before being washed and eluted as described above.

Western Blot Analysis

Proteins were separated on a 7.5% acrylamide gel, transferred onto nitrocellulose, and immunodetected with either an anti-phosphotyrosine antibody (4G10; Upstate Biotechnology, 0.1 µg/ml) or an anti-PRLR monoclonal antibody (U5 at 0.5 µg/ml) for an overnight period at 4 °C. Then, the membranes were incubated with a anti-mouse antibody coupled to peroxidase for 1 h at room temperature before being washed four times for 30 min in the washing buffer (50 mM Tris-HCl, pH 7.6, 200 mM NaCl, 0.05% Tween 20) and revealed by chemioluminescence (ECL kit from Amersham Corp.) according to the manufacturer's instructions. Membranes were stripped overnight at 4 °C in an acid solution (0.1 M glycine, pH 3, 0.1 M NaCl) and reprobed with an anti-Jak2 antibody (UBI, dilution 1/4000) and then with an anti-rabbit antibody coupled to peroxidase before being processed as described above.

Mutagenesis Experiments

The construction of the deletion mutants ( 243-267) and ( 296-322), as well as the truncation mutant (T322) were carried out using the Nb2 PRLR cDNA subcloned in M13 mp18 bacteriophage as a template. Single-stranded DNA was generated for the oligonucleotide-directed mutagenesis in RZ 1032 cells. After being checked by sequencing, the modified DNA were subcloned into the eukaryotic expression vector pR/CMV. The construction of the ( 268-287) mutant was carried out directly into the expression vector pR/CMV, using its M13 origin of replication and the MKO7 M13 helper phage. Single-stranded DNA was generated for mutagenesis reactions in CJ236 cells. Mutants were checked by DNA sequencing.

Prolactin-dependent Induction of -Casein Promotor/Luciferase Construct in 293 Cells

Cells were plated in 6-well plates (0.5 10 cells/well) before being transiently cotransfected as described before with 0.5 µg of pCH110 (-galactosidase expression vector, Pharmacia), 0.2 µg of the fusion gene carrying the promotor region of the rat -casein gene linked to the coding region of the luciferase gene and 0.1 µg of plasmid pR/CMV/long, pR/CMV/short, pR/CMV/Nb2, pR/CMV/ 243-267, pR/CMV/ 268-287, pR/CMV/ 296-322, and pR/CMV/T322. 1 day after transfection, cells were incubated in the presence or the absence of human PRL (5 10 M) and dexamethasone (250 nM) for 24 h before being lysed. Each value represents the stimulated luciferase activity measured in relative light units and normalized for -galactosidase activity. Results are the means ± S.E. of four different experiments.


RESULTS

Cotransfection of the cDNAs Encoding the Tyrosine Kinase Jak2 and the PRLR

To evaluate the association between the PRLR and Jak2, we have used transient expression in human 293 fibroblasts. Fig. 1shows the optimal conditions required for a clear induction of Jak2, and PRLR phosphorylation occurs with low but equal concentrations of each cDNA. Increasing the concentrations of cDNA for Jak2 results in its overexpression and nonspecific phosphorylation in the absence of ligand. Nevertheless, ligand-induced phosphorylation can still be observed, but the degree of induction is reduced. Increasing the amount of PRLR cDNA transfected also led to a high nonspecific signal in absence of prolactin. These results suggest that in 293 cells, it is extremely important to control the level of expression of both receptor and kinase to see clearly ligand-stimulated phosphorylation. If either receptor or kinase is overexpressed, autophosphorylation of both occurs in the absence of prolactin, perhaps due to an excess of kinase or to non-ligand-induced dimerization.


Figure 1: Specificity of ligand-induced phosphorylation of Jak2 and the PRLR. 293 fibroblasts (2.5 10/lane) were transfected with the indicated amounts of cDNAs encoding Jak2 and long form of PRLR. Prolactin receptors expressed at the cell surface were labeled with a monovalent Fab fragment generated from a monoclonal antibody (U5) directed against the extracellular domain of the PRLR, and the PRLR complexes were purified by incubation with anti-IgG-agarose beads. Proteins were separated on a 7.5% acrylamide gel, transferred onto nitrocellulose, and hybridized with an anti-phosphotyrosine antibody (4G10; Upstate Biotechnology) and revealed by ECL (Amersham), according to the manufacturer's instructions. The size of the two phosphorylated proteins, corresponding to the PRLR and the Jak2 kinase, are 95 and 130 kDa, respectively, and are indicated to the right.



Association and Activation of the Tyrosine Kinase Jak2 with the Different Natural Forms of the PRLR

The Nb2 form of the PRLR is a natural mutant of the long form of the receptor (Fig. 2) and has been shown, as well as the long form, to be constitutively associated with the tyrosine kinase Jak2 (18, 19, 20, 21) . We were interested in determining whether the short form was also able to bind and activate Jak2 as well as to undergo tyrosine phosphorylation itself. Fig. 3 , A and B, show that coexpression of the long or Nb2 forms of PRLR with Jak2 in human 293 fibroblasts results in association of the kinase with each form of PRLR and in tyrosine phosphorylation of both receptor and kinase. This indicates that PRLR binding and activation of the Jak2 kinase are key events in the transduction of the prolactin signal. Moreover, the deleted sequences within the cytoplasmic domain of the Nb2 form (Fig. 2) appear not to be necessary for Jak2 association. Interestingly, the short form was also able to bind and activate Jak2 but not undergo tyrosine phosphorylation, despite a high level of receptor expression (Fig. 3 C). This indicates that only the residues common to all three PRLR isoforms are required for Jak2 interaction. Moreover, our results also indicate that receptor phosphorylation occurs only in cytoplasmic regions present on PRLR long and Nb2 forms. This is especially important, since the short form of the PRLR is unable to generate a lactogenic signal (12, 13) nor to induce interferon regulatory factor-1 transcription (14) . This leads to the speculation that in addition to Jak2 phosphorylation, PRLR tyrosine phosphorylation is also required for prolactin signal transduction. An anti-Jak2 immunoblot was performed after cell surface labeling of the PRLR with a monovalent Fab fragment generated from an anti-PRLR monoclonal antibody (U5), as described in Fig. 1, and revealed no change in the amount of Jak2 bound to the short form of the PRLR in the absence or presence of prolactin (data not shown). This indicates that the short form of the PRLR is constitutively bound to the kinase Jak2, as is true for the long and Nb2 forms.


Figure 2: Schematic representation of wild type and mutant forms of PRLR. Illustrated are wild type forms of the rat long, Nb2, and short PRLR, as well as the mutant forms established from the Nb2 PRLR 243-267, 268-287, 296-267, and T322. Transmembrane domains are represented by black boxes. The juxtamembrane region conserved between GH and PRL receptors, containing the box 1 proline-rich motif, is stippled. Numbers to the right indicate the first amino acid, and the last amino acid of the mature protein. For the deletion mutants, the actual number is 393 minus the number of residues removed.




Figure 3: Association and activation of the tyrosine kinase Jak2 with the different forms of PRLR in response to prolactin. A, tyrosine phosphorylation of the kinase and the receptor. Human 293 fibroblasts were cotransfected with the calcium phosphate technique, with 2.5 µg of cDNA encoding the different natural forms of PRLR (long, short, or Nb2) and 2.5 µg of cDNA encoding the human tyrosine kinase Jak2. After stimulation by either the non-lactogenic biotinylated recombinant bovine GH or the lactogenic biotinylated oPRL, cell lysates were processed as described under ``Experimental Procedures'' and immunoblotted with a monoclonal anti-phosphotyrosine antibody. The position and the size of Jak2 (130 kDa) is indicated to the right as well as the size of the two forms of PRLR, which are tyrosine phosphorylated in response to prolactin, the long form (95 kDa), and the Nb2 form (62 kDa). B, Jak2 association with the different natural forms of the PRLR. The membrane described in A was stripped and reprobed with an anti-Jak2 antibody. The size of Jak2 is indicated on the right (130 kDa). C, to verify the correct expression of all the forms of PRLR, the membrane used in A was stripped and reprobed with a monoclonal anti-PRLR antibody. The size of the natural forms of the PRLR are 95, 62, and 42 kDa for the long, Nb2, and short forms, respectively.



Box 1 Is Sufficient for Jak2 Association and Activation

Since only box 1 is conserved in all three natural forms of the PRLR (Fig. 2), this led us to evaluate the importance of this region, as well as other regions of the cytoplasmic domain of the PRLR, for Jak2 association. We constructed PRLR mutants containing a deletion of 20 aa comprising the proline-rich domain ( 243-267), a deletion of 19 aa ( 268-287) corresponding to the region between the two boxes, or a control deletion of 27 aa, including part of the box 2 domain ( 296-322). Finally, we constructed a truncated mutant of the PRLR (T322), in which the carboxyl-terminal 70 aa, common to the long and Nb2 forms, were deleted (Fig. 2). Specific binding of I oPRL to microsomes prepared from transfected 293 cells, as well as whole cell binding, revealed that all wild and mutant forms of the receptor were expressed at approximately equal levels (data not shown). Immunoblot analysis of extracts from cells coexpressing the different mutants and Jak2 demonstrated that 268-287, 296-322, and T322 but not 243-267 were able to bind (Fig. 4, C and D) and induce tyrosine phosphorylation of Jak2 (Fig. 4, A and B). The cell surface labeling of these mutant forms expressed in 293 cells with the Fab fragment and the immunoblot analysis with an anti-Jak2 antibody, as described in the Fig. 1legend, also revealed that all the mutant forms of the PRLR, except 243-267, were constitutively bound to the kinase Jak2 (data not shown). These data, in addition to the previous results obtained with the short form of the PRLR, strongly suggest that the proline-rich region is the site of interaction with Jak2, implicating this sequence as being essential for signal transduction. Other parts of the juxtamembrane domain, as well as the carboxyl-terminal tail of the receptor, affect neither association of the receptor with Jak2 nor tyrosine phosphorylation of the kinase.


Figure 4: Association and activation of the tyrosine kinase Jak2 with different mutant forms of PRLR in response to prolactin. A and B, in this experiment, cells were cotransfected with the cDNA encoding Jak2 and one of the four mutant forms of the Nb2 PRLR (T322, 243-267, 296-322, and 268-287 compared with the wild type Nb2 PRLR). The immunoblot analysis was performed with an anti-phosphotyrosine antibody. The size of Jak2 (130 kDa) and the mutants 243-267 (59 kDa), 296-322 (58 kDa), 268-287 (59 kDa), and the wild type Nb2 PRLR are indicated to the right. C and D, Jak2 association with the mutant forms of the PRLR. The previous blots ( A and B) were stripped and reprobed with an anti-Jak2 antibody (UBI). E and F, the correct expression of the mutant forms of the PRLR was verified by reprobing of the membrane with a monoclonal antibody against PRLR. The sizes are 50, 58, 58, 59, and 62 kDa for T322, 243-267, 268-287, 296-322, and the wild type Nb2 PRLR, respectively.



We have previously shown in Nb2 cells that upon prolactin stimulation, the PRLR and Jak2 undergo tyrosine phosphorylation (19) . Examining receptor phosphorylation, shown in Fig. 4, A and B, indicates that the deletion mutant ( 243-267) lacking box 1 domain and the truncation mutant (T322) failed to induce receptor phosphorylation, while the deletion mutants 268-287 and 296-322 were able to undergo tyrosine phosphorylation themselves. These results further demonstrate that the site of interaction of Jak2 with the PRLR is mediated by box 1 only and that receptor phosphorylation requires the proper receptor-kinase association. The proper expression of these mutants has been verified by Western blot, as shown in Fig. 4, E and F. Since this proline-rich motif is found in many members of the cytokine receptor superfamily, it is likely that other members of the cytokine/GH/PRL family also interact with Jak2 through the same conserved domain (Fig. 5).


Figure 5: Amino acid sequences conserved in the cytoplasmic domains of members of the cytokine/GH/PRL receptor superfamily. The proline-rich region of the PRLR, GHR, IL-6R, gp 130, EPOR, granulocyte-colony stimulating factor receptor, and GM-CSFR subunits common to IL-3R, IL-5R, IL-2R, IL-3R, IL-4R, IL-7R, IL-5R, GM-CSFR, leukemia inhibitory factor receptor, and myeloproliferative leukemia virus receptor are shown. Amino acids highlighted in bold represent those identical to the PRLR. Numbers in parentheses indicate the beginning and end of the proline-rich region; the first amino acid (1) corresponds to the initiator methionine.



Regions of the Cytoplasmic Domain Required for Activation of -Casein Gene Promotor

To correlate these findings with the ability of the receptor to transmit a lactogenic signal, we cotransfected the different natural and mutant forms of the PRLR, described above, with the -casein promotor coupled to the luciferase gene in 293 cells, as described under ``Experimental Procedures.'' The results indicate that similar to the short form, the deletion mutants 243-267 and 268-287 as well as the truncation mutant T322 failed to activate the -casein promotor (Fig. 6). However, the deletion mutant 296-322 was fully capable of inducing -casein promotor to a similar level to that seen with PRLR long and Nb2 forms (Fig. 6). These observations indicate that three complementary mechanisms are needed for achieving full biological signaling of the PRLR. These include association of the kinase Jak2 with the PRLR mediated through box 1, the interbox region, as well as the carboxyl-terminal part of the receptor. The last two regions possibly present sites of interaction with other transducer molecules involved in prolactin signaling pathway.


Figure 6: Prolactin-dependent induction of -casein promotor/luciferase construct in 293 cells. Cells were transfected as described in under ``Experimental Procedures'' with the expression vectors containing the cDNAs encoding the different forms of PRLR. Results are expressed as the percentage of maximal activity (-fold induction). Results represent means ± S.E. of four independent experiments. The leftpanel represents the natural and mutant forms of the PRLR; the numbers to the right indicate the last amino acid of the mature protein. For the deletion mutants, the actual number is 393 minus the number of residues removed.



Activation of the Transcription Factor Stat1

Prolactin has been shown to activate two transcription factors belonging to the Stat family, Stat1 and Stat5. Stat1 has also been shown to be activated in response to different cytokines and to bind responsive elements related to the palindromic sequence recognized by Stat5. Stat5 activation has been shown to be indispensable for the hormonal induction of the -casein gene transcription. Therefore, we were interested in correlating the results of transcriptional activation described above with the ability of the different forms of PRLR to activate Stat1. For that purpose, we cotransfected a 293-cell line stably expressing the tyrosine kinase Jak2 with the cDNAs encoding Stat1 and a cDNA specific to the different forms of PRLR. As shown in Fig. 7 A, tyrosine phosphorylation of Stat1 is seen with all natural and mutant forms of PRLR, except 243-267, in which box 1 has been deleted. This suggests that activation of Stat1 depends on Jak2 and requires a functional kinase molecule bound to the receptor. In addition, activation of Stat1 seems to not require other regions of the cytoplasmic domain of the PRLR, since all other forms of the PRLR are able to induce Stat1 phosphorylation. When the membrane was stripped and reprobed with an antibody directed against Stat1, it is clear that the Stat protein is expressed equally in all samples (Fig. 7 B). The absence of clear induction in the phosphorylation of Stat1 upon prolactin stimulation is probably due to the high level of expression of the receptor, resulting in ligand-independent receptor dimerization and constitutive activation of the kinase Jak2. We confirmed this by cotransfecting the cDNAs encoding the long and short forms of the PRL receptor at different concentrations, with the cDNA encoding Stat1, in this cell line and obtained a clear prolactin induction of Stat1 phosphorylation by decreasing the amount of PRLR cDNA (data not shown). For Stat1 activation, Jak2 must be associated with the PRLR, as shown by the absence of signal when Stat1 is transfected alone in the stable cell line expressing Jak2. Since Stat1 activation is also mediated through cytokine receptors that are able to activate the tyrosine kinase Jak1, we examined the ability of the long form of the PRLR to induce tyrosine phosphorylation of Jak1 in our system. No Jak1 phosphorylation was observed following expression of PRLR in 293-LA cells (data not shown). This is in agreement with what we observed previously in Nb2 cells (19) .


Figure 7: A, activation of tyrosine phosphorylation of the transcription factor Stat1 with the different forms of PRLR in response to prolactin. The 293 stable cell line expressing the tyrosine kinase Jak2 was cotransfected with the cDNA encoding the different natural and mutant forms of PRLR and the cDNA encoding the Stat1 molecule. After stimulation by oPRL, cell lysates were immunoprecipitated with an anti-Stat1 antibody and immunoblotted with a monoclonal anti-phosphotyrosine antibody. The position and the size of Stat1 (91 kDa) and of the heavy chains of IgG (55 kDa) are indicated to the right. B, to verify the correct expression of Stat1 in all the assays, the membrane was stripped and immunodetected with an anti-Stat1 monoclonal antibody (Transduction Laboratories). The size of Stat1 (91 kDa) and of the heavy chains (55 kDa) are indicated to the right.




DISCUSSION

In this paper, we have examined different natural and mutant forms of the PRLR to determine regions of the receptor important for association and activation of the kinase Jak2, receptor phosphorylation, induction of the -casein gene promotor, and activation of the transcription factor Stat1. For this purpose, we developed a transient cotransfection system in which both the kinase Jak2 and the different forms of the PRLR were expressed in human kidney fibroblast 293 cell line. To see a clear result, it is important to determine the amount and ratio of each cDNA transfected. The fact that Jak2 is phosphorylated even in absence of prolactin stimulation could be due to an autoactivation of the kinase due of overexpression, but it also could reveal a normal state of phosphorylation, amplified in our system by the high number of copies of the kinase. In fact, it is clearly possible that some tyrosine residues of the kinase are phosphorylated, even in absence of stimulation, especially as the association between the kinase Jak2 and the PRLR has been shown to be constitutive (18, 19, 21) . Such phosphotyrosines, present in the kinase, could interact with the SH2 domain of an adaptor molecule, which in turn could interact with the receptor through another SH2 or an SH3 domain. Such a molecule, if it exists, remains to be identified.

Our studies reveal that the natural forms of the PRLR, including the short form, are able not only to associate with the kinase but also to activate its phosphorylation, suggesting that for this receptor, box 1 is sufficient for Jak2 association. To confirm this finding and extend it to the long and Nb2 forms of the PRLR, we prepared several deletion mutants of the cytoplasmic domain of the PRLR in the juxtamembrane domain and tested their ability to associate and activate the tyrosine kinase Jak2. Only the deletion of box 1 prevents Jak2 association with the PRLR. In contrast, a deletion between boxes 1 and 2 has no effect on the ability of the receptor to interact with the kinase, a result which differs to that obtained for EPOR and GM-CSFR (6, 7) . It is possible that association of Jak2 with some receptors involves different cytoplasmic regions, in addition to the conserved box 1 motif. Interestingly, box 1 resembles an SH3 binding site (2) , yet Jak2 does not contain an SH3 domain. This raises the possibility of an adapter molecule being required to interact with box 1 through a potential SH3 domain and bind Jak2 through a phosphotyrosine-SH2 domain interaction. However, this hypothesis remains to be verified and does not exclude other potential sites of interaction.

We observed PRLR tyrosine phosphorylation in Nb2 cells following prolactin stimulation (19) . Here, we demonstrate that activation of this process requires the membrane proximal region, box 1, and the membrane distal region common to the PRLR long and Nb2 forms. These results suggest that Jak2 activation is not sufficient to induce receptor phosphorylation. The carboxyl-terminal region of the receptor may possess sites capable of interacting with another protein tyrosine kinase. Alternatively, this carboxyl-terminal domain may harbor tyrosine residues that undergo phosphorylation in response to prolactin activation.

To correlate Jak2 association and activation and receptor phosphorylation with the biological activity of the PRLR, we developed a functional assay by coexpressing cDNAs encoding different forms of the PRLR with an expression vector containing the -casein promotor coupled to the luciferase gene. Utilizing this system, our results demonstrate that Jak2 association with the PRLR is necessary but not sufficient for activation of transcription. Thus, in addition to Jak2 association involving box 1, activation of milk protein gene transcription requires the presence of other residues in the membrane proximal region between boxes 1 and 2, as well as in the carboxyl-terminal part of the cytoplasmic domain. These results are in agreement with other observations, indicating that several regions, including box 1 and box 2, were required for the transmission of the growth signal by prolactin (5) .

Interestingly, the generation of mitogenic signals by other members of the superfamily of cytokine receptors seems to not require carboxyl-terminal domain sequences, as shown for the signal transducer gp 130 (22, 23) , granulocyte-colony stimulating factor receptor (24, 25) , IL-2R (26) , EPOR (27, 28, 29) , and the GHR (30) . However for the GHR, a recent report indicated that the carboxyl-terminal portion of the receptor is required for activation of transcription (31) . In addition, it has been shown that the carboxyl-terminal region of the cytoplasmic domain of the IFN was also necessary for activation of the IFN regulatory factor 1 gene (32) . For the long form of the PRLR from rabbit mammary gland, deletion of the carboxyl-terminal 70 aa has no effect on lactogenic signal transduction, but further deletion by another 70 aa into the region present in the Nb2 form results in loss of transcriptional signaling activity (33) . This suggests that the long form of the PRLR contains another site of interaction with the same or another transducer molecule of the prolactin signaling pathway and that the deletion of the carboxyl-terminal domain alone does not necessarily result in the loss the activity.

Transcriptional activation of cytokine and growth factor-responsive genes are mediated in part through tyrosine phosphorylation of Stat proteins. Stat1 is tyrosine phosphorylated in response to several cytokines including prolactin (3, 15) . Cotransfection of Stat1 cDNA with cDNAs encoding different forms of PRLR reveal that phosphorylation of Stat1 requires Jak2 association with the PRLR and does not appear to involve other cytoplasmic domains of the receptor. When increasing amounts of cDNAs encoding Jak2 and for Stat1 were transfected, a significant increase in -casein-luciferase activity was observed in the absence or presence of ligand but without changing the overall fold induction (data not shown). This indicates that the Jak2/Stat1 pathway may be involved in the -casein promotor regulation but is not sufficient for its activation. Since another member of the Stat family, Stat5, is also involved in the regulation of this promotor, it is possible that the complete and active transcription factor, which binds to the -casein promotor, could in fact be a homo- or heterodimer of Stat molecules, as it has been demonstrated for other receptors from the cytokine family. Tyrosine phosphorylation of Stat5 in response to prolactin has been shown to be mediated by the long form of the PRLR while the short form failed to transmit this effect (17) . This indicates that the mechanisms involved in activation of these two Stat proteins are different. It is possible that Stat1 activation is mediated through Jak2 activity, while Stat5 activation requires either direct interaction of the molecule with sequences present in the cytoplasmic domain of the long form of PRLR or indirect interaction, requiring the presence of other signaling molecules. Mutational analysis of the tyrosine residues present in Jak2 and PRLR are being carried out to investigate these possibilities.

Together, these results demonstrate that the tyrosine kinase Jak2 associates with the PRLR, a member of the cytokine/GH/PRL receptor family, through a proline-rich motif in the membrane proximal region, and that the interaction of the kinase with the receptor is necessary for activation of phosphorylation of the transcription factor Stat1 but not sufficient for signal transduction. Generation of a lactogenic signal further requires the association of the receptor with one or more signaling molecules within the cytoplasmic domain of the PRLR, which are likely to be necessary for coupling to specific signal-transducing proteins.


FOOTNOTES

*
This research was supported in part by grants from INSERM, the Fonds de Recherches en Santé du Québec, and the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Tel.: 331 40 61 53 10; Fax: 331 43 06 04 43.

The abbreviations used are: PRLR, prolactin receptor; oPRL, ovine prolactin; IL-2R, subunit of the interleukin-2 receptor; GHR, growth hormone receptor; GM-CSFR, granulocyte macrophage-colony-stimulating factor receptor; gp 130, -subunit of the IL-6R, leukemia inhibitory factor receptor; OSMR, oncostatin M receptor; CNTFR, ciliary neurotrophic factor receptor; EPOR, erythropoietin receptor; GM-CSFR, common -subunit of the GM-CSFR, IL-3R, and IL-5R; aa, amino acids; DMEM, Dulbecco's modified Eagle's medium.


ACKNOWLEDGEMENTS

We are grateful to Dr. J. Ihle for kindly providing the cDNA encoding for the tyrosine kinase Jak2, to Dr. R. Lammers for the antibody anti-Stat1 and for cloning the Stat1 cDNA, to Dr. J. Rosen for providing the rat -casein promoter, and to H. Buteau for preparing the -casein/luciferase construct, to Dr. J. Finidori for subcloning the Jak2 cDNA into the expression vector pCB6, to the National Hormone and Pituitary program for providing ovine prolactin, and to Dr. W. Brumbach for the recombinant bovine GH.


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