Dominant Negative and Cooperative Effects of Mutant Forms of Prolactin Receptor

Martine Perrot-Applanat, Oreste Gualillo, Alain Pezet, Valérie Vincent, Marc Edery and Paul A. Kelly

INSERM Unité 344 Endocrinologie Moléculaire Faculté de Médecine Necker 75730 Paris Cedex 15, France


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In addition to a long form of 591 amino acids (aa), two other forms of PRL receptor (PRLR), differing in the length of their cytoplasmic domains, have been identified in the rat. The Nb2 form, lacking 198 aa in the cytoplasmic domain, is able to transmit a lactogenic signal similar to the long form, whereas the short form of 291 aa is inactive. The ability of PRL to activate the promoter of the ß-casein gene or the lactogenic hormone responsive element fused to the luciferase reporter was assessed in Chinese hamster ovary cells or 293 fibroblasts transiently transfected with PRLR cDNAs. The function of the short form was examined after cotransfection of both the long and short forms. These results clearly show that the short form acts as a dominant negative inhibitor through the formation of inactive heterodimers, resulting in an inhibition of Janus kinase 2 (JAK2) activation. The present study also investigates the possible participation of cytoplasmic receptors in the signal transduction pathway, using cotransfection experiments and a new approach that selectively determines the contribution of cytoplasmic receptors in the process of signal transduction. We cotransfected Chinese hamster ovary cells with two cDNA constructs: a cytoplasmic (soluble) form of the receptor with a deleted signal peptide ({Delta}-19), which is unable to bind PRL, and a functionally inactive receptor mutant (lacking box 1), which is anchored in the plasma membrane and able to bind PRL. This approach has allowed us to show that {Delta}-19, lacking expression at the plasma membrane, can transduce the hormonal message, at least to a limited extent (up to 30% of wild type efficiency), providing that association/activation occurs with a PRL-PRLR complex initiated at the cell surface level; box 1 of the cytoplasmic form is necessary to rescue this partial transcriptional activity of the inactive mutant. This partial recovery is also parallel to the partial activation of JAK2, indicating that the signal transduction pathway implicated JAK2. Our results provide evidence that heterodimerization of receptors can be implicated either in the positive or in negative activation of gene transcription.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PRL is known to regulate a variety of physiological processes, including reproduction, mammary cell growth and differentiation, and immune functions (1), through its receptor (PRLR), which belongs to the superfamily known as hematopoietin/cytokine/GH-PRL receptors (2). This family includes the receptors for interleukins (IL-2 to IL-15), granulocyte-colony stimulating factor, granulocyte macrophage colony stimulating factor (GM-CSF), leukemia-inhibitory factor, ciliary neurotrophic factor, erythropoietin, GH, PRL, thrombopoietin , and leptin. After hormone binding, receptor dimerization, consisting of one molecule of PRL and two molecules of receptor, appears to be the first step by which PRL mediates its various actions (3). The activation of the receptor, which does not possess an intrinsic tyrosine kinase, leads to the rapid phosphorylation of an associated cytoplasmic tyrosine kinase, Janus kinase 2 (JAK2), which is necessary for the phosphorylation of the receptor and signal transduction molecules (for review, see Refs. 4, 5, 6); box 1 (a proline-rich motif) and the adjacent residues upstream of box 2 in the cytoplasmic proximal region (near the membrane-spanning domain) of cytokine receptors are sufficient for JAK kinase association/activation (7, 8). The major downstream targets of PRL-activated JAK kinases include members of the Shc-Ras pathway (9) and some members of the signal transducers and activators of transcription (STAT) protein family (10), including STAT1, STAT3, and STAT5 or MGF (mammary gland factor) (11, 12). Members of the STAT family that possess a SH2 domain require tyrosine phosphorylation for activation. In addition to a long form of 591 amino acids (aa), two other forms of PRLR differing in the length of their cytoplasmic domains have been identified in the rat. As is true for the long form, the Nb2 form, lacking 198 aa in the cytoplasmic domain, is able to transmit the lactogenic signal, while a short form of 291 aa is inactive in lactogenic bioassays (13, 14, 15, 16). In the first part of this study, we investigated the effect of the short form of rat PRLR on the activity of the long form of PRLR in differentiated cells.

Signal transduction through cytokine receptors, as for receptor tyrosine kinases, is believed to occur almost exclusively at the level of the plasma membrane. Several growth factors [epidermal growth factor (EGF), insulin], as well as PRL, induce the rapid internalization of their receptors by hormonally responsive cells using a receptor-mediated process (17, 18, 19, 20, 21). PRL and PRLR endocytosis and recycling remain poorly understood, as compared with other membrane receptors (such as insulin, EGF, low-density lipoprotein, transferrin; see Refs. 22, 23 for a review). Rapid internalization of growth factors [EGF, platelet-derived growth factor (PDGF)] and their receptors, which belong to the receptor tyrosine kinase family, into endosomes is traditionally thought to attenuate the ligand-induced response; however, there is also growing evidence for selective and regulated signal transduction occurring within the endosome, based on the presence of an active growth factor tyrosine kinase and tyrosine-phosphorylated signaling molecules. In fact, there is a paucity of data to associate receptors and signaling pathways to membrane trafficking, including the first step, endocytosis. For several growth factors (EGF, PDGF), both the mechanism of endocytosis and signal transduction require the phosphorylation of tyrosine residues by the kinase domain located in the cytoplasmic region of the receptor (22, 23). Internalization and signal transduction by the GH receptor are mediated by different regions of the cytoplasmic domain (24). Similar studies concerning internalization of the PRLR are currently in progress, and for other cytokine receptors, the requirement of a tyrosine kinase being directly required for internalization remains unknown. In the second part of this study, we have investigated the possible implication of cytoplasmic receptor constructs in signal transduction.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PRLR Short Form Is a Dominant Negative Inhibitor of the Long Form of Receptor
The different forms of the rat PRLR, differing in the length of their cytoplasmic domains (13), were efficiently expressed when transfected in COS-7 cells, as judged by their PRL-binding capacity and affinity and immunolocalization using specific anti-PRLR antibodies (Table 1Go). To analyze the influence of the expression of the short form of PRLR upon transcriptional activity of the long form, we used a functional transcription assay, which consists of cotransfecting a ß-casein/luciferase reporter gene construct with PRLR cDNA (long form, short form, or mixture of long and short forms). Initial studies were performed in Chinese hamster ovary (CHO) cells, which have no detectable endogenous PRLR and are thus a suitable model system for these studies. As shown in Fig. 1AGo, the long form of PRLR, in contrast to the short form, activated the ß-casein/luciferase reporter gene upon PRL stimulation. This functional activation of the long form of PRLR was reduced in the presence of the short form of PRLR. The ß-casein/luciferase induction progressively decreased in the presence of increasing amounts of the short form of PRL-R. This competition of the short form PRLR with the activity of the long form was observed at several concentrations of ovine (o)PRL (400 ng to 10 µg/ml) (not shown). Similar results have been obtained with a bovine mammary epithelial cell line (BGME) cotransfected with long and short forms of rat PRLR cDNA (not shown).


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Table 1. Characteristics of Wild Type and Mutant PRLRs

 


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Figure 1. Inhibition of the Long Form of PRLR Activity by the Short Form Receptor

A, CHO cells were cotransfected with a construct that contains a milk protein (ß-casein) gene promoter fused to the reporter gene of the firefly luciferase (plasmid-casein/luciferase) and PRLR cDNA, as described in Materials and Methods. After incubation with oPRL (400 ng/ml) and dexamethasone (250 nM) for 48 h at 37C, cells were assayed for luciferase activity. Cotransfections experiments included the transfection of either the long form or the short form of PRLR cDNA alone (long 6:0 or short 0:6, respectively), or a mixture of both the long and short forms of PRLR cDNAs. Control long or short form/luciferase-transfected cells had a basal luciferase activity (without hormone) of 500 RLU/U ßgal. In these experiments, results of luciferase activity were assayed in cells transfected with several ratios (6:0 to 0:6, for 6 µg total cDNA) of long and short forms of PRLR cDNA. Results of luciferase activity of ß-casein gene promoter upon PRL stimulation (400 ng/ml) are expressed as fold-induction compared with cells not stimulated with PRL. Results represents the means ± SEM of three to five independent experiments. B, Same experiments in 293 cells using LHRE fused to the reporter gene of firefly luciferase. Results of luciferase activity were assayed in cells transfected with several ratios (1:0 to 1:5, for 6 µg total cDNA) of long and short forms of PRLR cDNA.

 
To study in detail the dominant negative action of the short form, further studies were conducted in the human embryonic kidney fibroblast 293 cell line in which both functional assay and Western blot analysis are possible due to the high level of expression of plasmids under the control of the CMV promoter in this cell line. As shown in Fig. 1BGo, in cotransfection experiments, increasing concentration of the short form of PRLR resulted in a stepwise decrease in the functional activity of the long form of PRLR. Luciferase fold induction using the ß-casein promoter was routinely in the range of 5–7, whereas it was much higher using the lactogenic hormone response element (LHRE) construct (see Materials and Methods) for which we obtained maximal activation of 18-fold for the long form alone. Again, a drastic inhibition (90%) was obtained with a 1:5 ratio of long to short form of PRLR cDNA, similar to what was obtained in CHO (Fig. 1Go) or BMGE cells, (J. J. Berlanga, J. P. Garcia-Ruiz, M. Perrot-Applanat, P. A. Kelly, and M. Edery, manuscript submitted). The same results were obtained when the intermediate Nb2 form was used instead of the long form of the PRLR (data not shown).

Mechanisms of the Dominant Negative Action of the Short-Form PRLR
To investigate how this dominant negative action of the short form occurs, two series of experiments were conducted. In the first set of experiments, we analyzed by Western blots the activation of JAK2, which is known as the primary kinase activated during PRL signaling.

In 293 cells cotransfected with long and short forms of the PRLR cDNA, there was a drastic reduction in the activation of JAK2 upon PRL stimulation, as shown by Western blot revealed with antiphosphotyrosine antibodies (Fig. 2AGo, lanes 3–4), whereas cells transfected with the long form only displayed a clear activation of JAK2 upon PRL stimulation (Fig. 2AGo, lanes 1 and 2). Similar results were obtained with cotransfection of Nb2 and short forms of PRLR cDNA (Fig. 2AGo, lanes 5–8). Control experiments in which blots were revealed using JAK2 antibody indicate a similar level of JAK2 expression in cells stimulated or not by PRL (see Fig. 2BGo). As has been previously described (8), more JAK2 was associated with the Nb2 form of PRLR (Fig. 2AGo, lanes 5 and 7). As is common in transient transfections, some slight differences in the amount of JAK2 expressed was observed (Fig. 2AGo, lanes 3, 5, and 7). Also, the level of tyrosine phosphorylation of PRLR was greatly reduced when the short and long forms of PRLR were cotransfected (data not shown). Thus, the short form of the rat PRLR exerts a dominant negative effect on the activation of the long-form receptor, via the inhibition of the activation of JAK2 kinase.



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Figure 2. Inhibition of the Activation of JAK2 by the Short Form of PRLR

293 cells (2.5 x 106/lane) were cotransfected with 1 µg cDNA encoding the long form of PRLR alone (control, lanes 1–2) or together with 5 µg cDNA encoding the short form of PRLR (lanes 3–4); with 1 µg cDNA encoding the Nb2 form of PRLR alone (lanes 5–6); or together with 5 µg cDNA encoding the short form of PRLR (lanes 7–8); and with 0.5 µg of cDNA encoding JAK2. After stimulation (+) or not (-), cell lysates were processed as described in Materials and Methods and immunoprecipitated with an anti-JAK2 monoclonal antibody. Immunoprecipitates were resolved on 7% SDS-PAGE, and the blots were probed with antiphosphotyrosine antibody (panel A) or anti-JAK2 antibody (panel B).

 
In the second set of experiments, we investigated whether the dominant negative effect of the short form resulted from the formation of heterodimers between long and short forms of PRLR. For this purpose, we generated a cDNA of the long form of the rat PRLR containing an oligonucleotide encoding a Flag epitope inserted just before the N-terminal amino-acid (named Flag-tagged PRLR); the encoded protein is specifically recognized by anti-Flag antibodies (25, 26). Complementary DNA encoding Flag-tagged PRLR long form was cotransfected with the nontagged short form cDNA in 293 cells, proteins were immunoprecipitated using anti-Flag antibody and further analyzed by Western blot using either anti-Flag antibody or anti-PRLR (U5). When blots were analyzed with anti-Flag antibody, a single band appeared in cells cotransfected with both PRLR cDNAs and in control cells transfected with PRLR long form alone; this band correspond to the Flag-PRLR (95 kDa) (Fig. 3Go, lanes 1–4). On the other hand, when monoclonal anti-PRLR antibody (U5) was used for blotting, two bands of 95 kDa and 42 kDa were revealed in cotransfected cells upon PRL stimulation (Fig. 3Go). In control cells transfected with Flag-PRLR alone or in control cells cotransfected in the absence of PRL, only one band (95 kDa) was observed. The two bands present in cotransfected cells upon PRL stimulation correspond to the long and short forms of PRLR, indicating that the Flag antibody, which is specific for the long form of PRLR, was able to coimmunoprecipitate long and short forms of PRLR resulting from heterodimerization.



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Figure 3. Heterodimerization of Short and Long Forms of PRLR

293 cells were cotransfected with 1 µg cDNA encoding a Flag-tagged long form of PRLR(L) alone (lanes 1–2 and 5–6) or together with 5 µg cDNA encoding the short form of PRLR(S) (lanes 3–4 and 7–8). Cell lysates were processed as described in Materials and Methods and immunoprecipitated with an anti-Flag monoclonal antibody. Immunoprecipitates were resolved on 8% SDS-PAGE, and the blots were probed with an anti-Flag monoclonal antibody (lanes 1–4) or an anti-PRLR monoclonal antibody (U5) (lanes 5–8).

 
Synthesis of Inactive Mutants of the PRLR Expressed at the Cell Surface
We constructed mutants of PRLR (Fig. 4AGo), which are inactive in the ß-casein/luciferase reporter assay when expressed in CHO cells, but retain the characteristics of the wild type receptors with respect to their expression at the cell surface, binding affinity, and endocytosis (Table 1Go). A mutant of the Nb2 form of PRLR in which the four prolines in box 1 are replaced by alanine (4P/A) has no activity due to its lack of association and activation of JAK2 (8). As shown in Fig. 4BGo and Table 1Go, mutations in the box 1 region in the PRLR long form (4P/A and P250L, see Materials and Methods) resulted in the loss of ability to transduce the PRL signal, but neither binding nor ability to internalize of the mutants was altered (see Fig. 4CGo). Also, the level of expression of these mutants was similar to that of the wild type receptor, as evaluated using Western blots (Fig. 4DGo).



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Figure 4. Functional Expression of Wild Type and Mutants PRLRs

A, Schematic representation of wild type and mutant PRLR: long form, P250L (substitution of proline 250 to leucine), 4P/A (substitution of prolines 245, 246, 248 and 250 to alanine), {Delta}-19 (deletion of the signal peptide), {Delta}-19, 4P/A (deletion of the signal peptide and substitution of prolines 245, 246, 248, and 250 to alanine), NLS (addition of a nuclear localization signal), short form, Nb2 form and Nb2-T322 (truncation of the cytoplasmic tail of the Nb2 form). Generation of the mutants via a recombinant PCR strategy is described in Materials and Methods. B, Stimulation of functional activity by PRL. Cells were cotransfected with ß-casein/luciferase construct and PRLR cDNAs and assayed for luciferase activity after stimulation with (or without) oPRL (400 ng/ml, 48 h) as described in Materials and Methods. Results are expressed as fold-induction of luciferase activity in the presence of PRL, as compared with the activity in the absence of the hormone. Mutation of box 1 or deletion of the signal peptide of PRLR leads to inactive PRLRs. Values represent means ± SEM of five independent experiments. C, Receptor-mediated endocytosis of wild type and mutants PRLR in COS-7 cells. Cells were transfected with PRLR cDNA wild type (long), Nb2, 4P/A, or {Delta}-19 + 4P/A mutant form of the PRLR and incubated with [125I]hGH. The kinetics of internalization were determined after different periods of incubation, as described in Materials and Methods. Kinetics of internalization of P250L mutant is similar to that of 4P/A mutant. These results are representative of three independent experiments performed in duplicate. D, Expression of different mutant forms of the PRLR. 293 cells were transfected with 2 µg cDNA encoding the PRLR wild type (WT), the P250L, the 4P/A, and the {Delta}-19 mutant forms of the PRLR. Cell lysates were processed as described in Materials and Methods and immunoprecipitated with a monoclonal antibody (U5) directed against the extracellular domain of the PRLR, except for the {Delta}-19, which was immunoprecipitated with an anti-Flag monoclonal antibody M2 ({alpha} Flag). Immunoprecipitates were separated using an 8% SDS-PAGE, blotted onto PVDF membranes and then probed with an anti-PRLR antibody (U5) for WT, P250L, and 4P/A or an anti-Flag antibody for {Delta}-19.

 
Synthesis of Inactive PRLR Mutants Expressed in the Cytoplasm (Soluble Mutants)
We next constructed a cytoplasmic (soluble) form of the PRLR, by deleting the sequence encoding the signal peptide ({Delta}-19) (Fig. 4AGo) (see Materials and Methods), which resulted in a mutant receptor unable to enter the rough endoplasmic reticulum during the biosynthetic pathway and thus, to be targeted to the plasma membrane. Immunofluorescence analysis confirmed its cytoplasmic localization, with a diffuse pattern clearly different from the immunofluorescence pattern corresponding to the normal protein secretory pathway (RER, Golgi apparatus, and vesicles as shown in Fig. 5Go and in Ref. 25). This mutant receptor was functionally inactive, as judged by the absence of PRL binding and ß-casein/luciferase transcriptional activity in the presence of PRL (Fig. 4BGo). Also, the receptor lacking the signal peptide was equally stable compared with the wild type receptor, as observed by Western blot (a single band of 85 kDa), and immunofluorescence after 0–12 h PRL stimulation (Fig. 4DGo and Fig. 5Go). Characteristics of the various mutant PRLRs are summarized in Table 1Go.



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Figure 5. Immunofluorescence Localization of PRLR or {Delta}-19 Mutant Receptor

COS-7 cells were transfected with cDNAs encoding PRLR (a, b, and c) or {Delta}-19 mutant cDNA (d) and processed for indirect immunofluorescence, as described in Materials and Methods. PRLR was expressed at the cell surface (c, as observed in non permeabilized cells) and in membranous compartments (probably rough endoplasmic reticulum, Golgi area, and vesicles as observed in permeabilized cells; see the Golgi area and vesicles in panel a); in contrast, {Delta}-19 mutant receptor is expressed within the cytoplasm as a soluble protein (d), as deduced from the diffuse pattern characteristic of cytoplasmic protein (50 ). b, Phase contrast. Magnification, x300. The expression of the {Delta}-19 mutant receptor is stable over a 12-h stimulation (25 ).

 
Soluble {Delta}-19 PRLR Mutant Rescues Membrane-Inactive Box 1 PRLR Mutant
When CHO cells were cotransfected with a PRLR cDNA encoding a box 1 inactive mutant (4P/A or P250L) and a cDNA encoding a cytoplasmic form of the receptor ({Delta}-19) along with the ß-casein/luciferase reporter gene, transcriptional activity was significantly (P < 0.05) recovered upon PRL stimulation (400 ng/ml) up to 28 ± 3% of wild type efficiency (Fig. 6Go), suggesting a cooperative effect of the two mutant forms of receptor. This increase was PRL-dependent inasmuch as basal luciferase activity of the two cotransfected mutants was not modified in the absence of hormone, as compared with basal activity of each mutant alone. Similar results were observed at various concentrations of oPRL (400 ng/ml to 10 µg/ml) (not shown). Maximal recovery was obtained with similar microgram amounts of cotransfected cDNAs (3:3 to 4:2 ratios of cDNAs of cytoplasmic vs. plasma membrane mutants). These results suggest that some functional hormone-dependent interaction may occur between an inactive membrane-bound PRLR and a cytoplasmic mutant.



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Figure 6. Rescue of an Inactive PRLR (Long Form) Expressed at the Cell Surface by a Cytoplasmic PRLR Mutant

Cells were cotransfected with ß-casein/luciferase construct and a cDNA mixture of an inactive box 1-mutated PRLR (4P/A or P250L) and a cytoplasmic PRLR ({Delta}-19). Luciferase activity was assayed after stimulation with oPRL (400 ng/ml for 48 h), as described in Materials and Methods. Control experiments included transfection with the long form of PRLR, or P250L, 4P/A, {Delta}-19 or {Delta}-19, 4P/A mutant cDNA alone. Basal luciferase activity (in the absence of PRL) of each mutant alone was not modified by the presence of another mutant in cotransfection experiments. In the experiment shown in this figure, results of luciferase activity were assayed in cells transfected with a 1:1 ratio of cDNAs (corresponding to 3 µg cDNA for each mutant). Cells were also cotransfected with a cDNA of an inactive box 1-mutated PRLR (4P/A or P250L) and a cytoplasmic/box 1-mutated PRLR ({Delta}-19, 4P/A). Values represent means ± SEM of five independent experiments performed in duplicate.

 
As the box 1 region is known to be necessary for signal transduction (8, 27), we investigated the possible involvement of box 1 of the cytoplasmic mutant on the partial recovery of transcriptional activity. Cotransfection experiments using PRLR cDNAs encoding an inactive cell surface receptor and a cytoplasmic mutant, both receptors being mutated in box 1 (mutants 4P/A or P250L and {Delta}-19, 4P/A, see Fig. 4Go, A and B), failed to activate the ß-casein/luciferase reporter gene (Fig. 6Go). This suggests that an intact box 1 of the cytoplasmic form of the receptor is necessary for the recovery of partial transcriptional activity, after PRL stimulation.

In contrast, no functional activity could be recovered when the inactive short form of PRLR or the truncated mutant T322 of the Nb2 form (see Fig. 4BGo) was cotransfected with the {Delta}-19 (Fig. 7Go), although these forms are binding forms and are able to internalize (Table 1Go). These results probably reflect the requirement for distal C-terminal sequences in the PRLR cytoplasmic domain (in addition to the box 1 region) for the partial recovery of transcriptional activity. Association of the two receptor forms probably occurs in the cytoplasm because cotransfection experiments with a nuclear mutant, generated by addition of a nuclear localization sequence (NLS) in the ({Delta}-19) PRLR mutant (see Fig. 4AGo), and an inactive box 1-mutated PRLR did not permit recovery of transcriptional activity of the inactive mutant (long form) expressed at the plasma membrane (Fig. 7Go). This further confirms that the partial recovery of transcriptional activity of the two inactive mutants is only observed for receptor interactions occurring in the cytoplasm and in the presence of PRL.



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Figure 7. Inactive Short and Mutant Nb2 Receptors Are Unable to Be Rescued by a Cytoplasmic PRLR Mutant

Cells were transfected with ß-casein/luciferase construct and cDNAs of the short or mutant Nb2 T322 and {Delta}-19. Control experiments included transfection with cDNAs encoding the long form, {Delta}-19, Nb2 T322, or NLS+4P/A. Values represent the means ± SEM of three independent experiments performed in duplicate.

 
To determine whether the partial activation of transcription observed after cotransfection of {Delta}-19 and 4P/A resulted from a partial activation of the JAK/STAT pathway, the activation of JAK2 was determined as described before in 293 cells (8). Briefly, 293 cells were cotransfected with {Delta}-19, and 4P/A and JAK2 cDNAs, immunoprecipitated with anti-PRLR monoclonal antibody, and further analyzed by Western blot using antiphosphotyrosine antibody. Whereas the transfection of the long form of PRLR used as control resulted, as previously described (8), in the phosphorylation of JAK2 (130 kDa) and PRLR (95 kDa), the cotransfection of {Delta}-19 and 4P/A resulted in a reduced, albeit still significant, phosphorylation of JAK2 and PRLR after PRL stimulation (Fig. 8Go).



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Figure 8. Rescue of an Inactive PRLR (Long Form) by a Cytoplasmic PRLR Mutant involves JAK2 Activation

293 cells were transfected with 2 µg cDNA encoding the long form of the PRLR (lanes 1–2) or with cDNAs encoding the 4P/A and {Delta}-19 mutant forms (lanes 3–4), along with 0.5 µg cDNA encoding JAK2. After stimulation (+) or not (minus]) with 400 ng/ml oPRL (18 nM), cell lysates were immunoprecipitated with a monoclonal antibody (U5) against the PRLR. Immunoprecipitates were separated using a 8% SDS-PAGE, blotted onto PVDF membranes, and then probed with an antiphosphostyrosine antibody. The positions of JAK2 (130 kDa) and PRLR (95 kDa) are indicated on the ight.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Since the cloning of PRLR cDNAs revealed their existence in multiple forms (i.e. long, short, and intermediate) (13), extensive studies on the function of each form have been reported. Using CHO cells cotransfected with PRLR cDNA and a reporter gene (CAT or luciferase) under the control of a milk protein gene promoter, it has previously been shown that both the long and intermediate forms of the rat PRLR were able to induce expression of the reporter gene (14, 15, 16), whereas the short form was inactive (14). On the other hand, both the long and short forms of the PRLR were shown to transmit a mitogenic signal in transfected NIH 3T3 cells (15). Using cotransfection experiments in CHO and 293 cells, the present study demonstrates one additional function of the short form of PRLR in PRL signaling of milk protein gene activation: that it can act as a negative regulator, supporting a novel view in the understanding of PRL action. Since the first step in PRL action is the dimerization of the receptor induced by PRL (3), the composition of PRLR-PRL complexes is fundamental to the ability of PRL to stimulate milk protein gene transcription. The homodimerization of PRLR allows proper docking of transducing molecules both at the proline-rich region (box 1), such as JAK2 (8), and at a C-terminal tyrosine (28). This tyrosine has been shown to be responsible for activation of gene transcription in the Nb2 PRLR and to be the major regulator in the long form of PRLR (28). It has been suggested that this phosphorylated tyrosine could be involved in association and activation of STAT5 (12); the region surrounding this residue may contain an interaction site for the tyrosine phosphatase PTP1D (29).

When the short form is coexpressed with the long form of the PRLR, PRL signaling to milk protein genes is blocked in a dose-dependent manner. This inhibitory activity of the short form of PRLR could reflect the sequestration of PRL by an increase of inactive homodimers of short form or that heterodimerization of long and short forms of PRLR prevents PRL signaling (nonfunctional heterodimers). The dominant negative effect of the short form is further supported by the fact that JAK2 activation, as well as PRLR phosphorylation, which represent the earliest events in PRLR activation after PRL binding, were inhibited after cotransfection experiments of long and short forms. Evidence for the formation of heterodimers between long and short forms of PRLR is given from cotransfection experiments using a Flag-tagged long form PRL cDNA and a nontagged short-form PRLR cDNA (Fig. 3Go). This dominant negative effect of the short form implies that the STAT protein needs a pair of distal receptor domains, presumably the two distal phosphotyrosines, for proper docking, and that homodimerization of receptors is required for proper signaling. Further experiments involving specific phenylalanine mutations are required to correlate receptor phosphorylation and biological activity. Finally, the ratio of expression of the short to the long form of PRLR has been shown to vary in a tissue-specific manner and also as a function of physiological state, providing cells a powerful means of modulating PRL action. A clear example is illustrated by the mammary gland and liver, in which reverse ratios of long and short forms exist (30). A similar mechanism of modulation of cell signaling through formation of homo- and heterodimers has been seen for insulin, erythropoietin, TNF{alpha}, and Fas/APO1 effects (31, 32, 33). However, whether PRL-(Long PRLR-Short PRLR) or PRL-(Short PRLR)2 complexes are involved in other functions of PRL is still an open question. It is interesting to note that, using GM-CSF receptor/PRLR chimera, it has been shown that Nb2PRLR-Short PRLR heterodimers failed to induce proliferation in BAF3 line, and this was related to an absence of activation of JAK2 and Fyn, whereas the GM-CSF receptor/Nb2PRLR homodimers are fully capable of GM-CSF-induced proliferation and activation of JAK2 and Fyn in the BAF3 transfectants (34). Together, these data and the present findings indicate that the Short PRLR functions as a dominant negative isoform, inhibiting both proliferation and activation of milk protein gene transcription by the receptor complex through heterodimerization (Fig. 9Go). Indeed, such heterodimers are shown to occur in human embryonic kidney fibroblast 293 cells.



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Figure 9. Hypothetical Model of Inactive and Active PRLR Heterodimers

A, Binding of PRL to its receptor induces heterodimerization. JAK2 is associated to the PRLR through the box 1 sequence (black box) in the cytoplasmic domain, but there is no activation of JAK2 or of intracellular PRLR tyrosines. B, Binding of PRL to its receptor induces PRLR dimerization. Recruitment of cytoplasmic box 1 active PRLR results in transphosphorylation (dashed arrow) of JAK2 itself as well as the PRLR intracellular tyrosines that provide docking sites for Stat5 association and activation leading to signal transduction.

 
We also investigated the possible implication of cytoplasmic receptors in signal transduction of PRLR, as such soluble receptors have been reported (35). For this purpose, we developed a new approach that selectively determines the contribution of cytoplasmic receptor constructs in the process of signal transduction. We cotransfected CHO cells with two cDNA constructs: {Delta}-19 and 4P/A or P250L, the last two encoding receptors anchored in the plasma membrane and able to bind PRL. The {Delta}-19 receptor is not processed through the endoplasmic reticulum and the Golgi apparatus where the formation of disulfide bonds and glycosylation occurs and does not bind PRL even when a cytosolic preparation is used (Table 1Go), probably due to an improperly folded extracellular domain. This approach has allowed us to show, for the first time, that a mutant of the PRLR, lacking expression at the cell surface can transduce the hormonal message, at least to a limited extent (up to 30% of wild type efficiency), providing cooperation via association/activation occurs with a PRL-PRLR complex initiated at the cell surface. The rescue and activation of a binding deficient insulin receptor has been previously reported by a membrane-adjacent receptor (36). That only partial recovery of transcriptional activity was observed in our system can be explained by the fact that we used a soluble receptor designed only to detect a potential cytoplasmic activation pathway. Assuming that 100% functional activity corresponds to the combination of plasma membrane and cytoplasmic contributions, 30% recovery of activity observed in this study could represent the maximal contribution of intracellular receptors or could be reflective of the maximal activation possible when only a subset of {Delta}-19, that portion which is in the membrane-proximal region of the cytoplasm, is available to associate with the box 1 mutant receptor. In addition, partial recovery also could be due to the stoichiometry involving {Delta}-19 and the inactive membrane-bound mutants, which might limit the potential formation of tetramers and oligomers.

The fact that box 1 of {Delta}-19 is necessary for the partial transcriptional activity of the 4P/A mutant expressed at the plasma membrane suggests that the biological responses observed in this study probably occur through box 1, as is true for wild type and Nb2 PRLRs (4, 5, 28). This partial recovery is also parallel to the partial activation of JAK2 and phosphorylation of the PRLR after cotransfection and PRL stimulation, indicating that the signal transduction pathway implicates JAK2 and box 1 of PRLR. The lack of recovery of milk protein gene transcription with Nb2 T322 or the short form suggests that other regions of the cytoplasmic domain, in addition to box 1, are involved in activation. Indeed, cytoplasmic segments of PRLR have been shown to provide cooperating or conflicting intracellular signals (12). Such a negative domain has been shown for the erythropoietin receptor, which contains a site of interaction for the tyrosine phosphatase PTP1C or SHP1 (37). Also, the fact that box 1 of {Delta}-19 and the full-length cytoplasmic tail of 4P/A or P250L mutant must both be present for partial recovery of transcriptional activity support the concept of heterodimerization and transphosphorylation of PRLRs, as has also been described for the insulin receptor (38) (Fig. 9Go). Partial activation was obtained using an inactive box 1 PRLR mutant for which endocytosis after PRL stimulation is not altered. That PRL interacts with its receptor intracellularly under normal conditions has been previously described in the 235–1 mammotroph PRL-secreting cell line (39); furthermore, cytosolic PRLRs have been identified (35); and PDGF receptor activation in intracellular compartments by an autocrine mechanism has been described in v-sis-transformed cells (40). Further experiments using mutagenesis of the soluble form of the PRLR are needed to determine the interaction domain(s) in the cytoplasmic tail of the two receptors.

This study also provides evidence that box 1, a proline-rich region, is not required for internalization of the PRL-PRLR complex. The fact that box 1 is required for signal transduction, but not for endocytosis, contrasts with the situation observed for several growth factor receptors, including those for EGF and PDGF, for which phosphorylation of tyrosine residues by the kinase domain is required (22). Recently, it has been shown that a mutant EGF receptor lacking a functional tyrosine kinase cannot undergo efficient recruitment into coated pits (41). However, it is not impossible that a PRLR-associated kinase, distinct from JAK2, could be required for the PRLR internalization. In addition, the domains required for internalization and signal transduction may be located in different regions of the cytoplasmic domain of the PRLR.

Our experiments, based on a new approach that selectively determines the contribution of cytoplasmic receptor, suggest that the association of the two inactive mutants can occur at the cytoplasmic face of the cell membrane, or anywhere in the cytoplasm where 1) the soluble form is present in high amounts, 2) the membrane form is endocytosed, and 3) all necessary signal-transducing molecules are present and can associate with such receptor. In the latter case, the process of signal transduction generated by an activated PRLR at the cell surface can be prolonged inside the cell through heterodimerization. However, experiments using a PRLR box 1 mutant, which also cannot be endocytosed, are necessary before conclusions can be made about whether the partial rescue of transactivation by the cytoplasmic PRLR rely on endocytosis. Although signal transduction through cytokine receptors, as is true for tyrosine kinase receptors, is believed to occur almost exclusively at the level of the plasma membrane, a model in which endocytosis plays a role in transmitting a ligand-dependent signal has been proposed on theoretical grounds (22, 23, 42): 1) rapid internalization of the growth factor and its receptor, and 2) presence of an active growth factor tyrosine kinase and tyrosine-phosphorylated signaling molecules within the endosome.

Thus, heterodimerization of the PRLR can result either in an inhibition or an activation of gene transcription, depending of the formation of active or inactive dimers, which presents an alternative in modulating PRL action. A model that depicts these two mechanisms is shown in Fig. 9Go.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PRLR and Mutants
The full-length cDNA for the long, short, and Nb2 forms (16, 43) was used for these studies. Several mutants of the PRLR were generated from the long form and the Nb2 form of the rat PRLR via a recombinant PCR strategy, as follows.

The construction of P250L and 4P/A mutants was generated in the pR/CMV vector (Invitrogen, San Diego, CA) as a template, by substitution of proline 250 to leucine and prolines 245, 246, 248, and 250 to alanine, respectively.

Deletion of the signal peptide (19 aa) of PRLR alone and deletion of the signal peptide along with mutation of 4P/A of PRLR resulted in {Delta}-19 long form and {Delta}-19,4P/A PRL-R mutants, respectively (cytoplasmic forms). Deletion of the signal peptide of PRL-R (mutant {Delta}-19) was generated using the cDNA encoding the long form of rat PRL-R in Bluescript expression vector with PCR amplification of PRL-R cDNA, in the presence of a 5'-oligonucleotide primer containing an EcoRI site, the codons for an initiation methionine, and the first five aa after the signal peptide (aa 20–24: QSPPG, see Ref. 43 for the abbreviation) of wild type PRLR (5'-GTTAACGAATTCATGCAGTCACCACCAGGG-3'); long sense oligonucleotide contained a sequence complementary to the region encoding amino acids 205–210 and an NcoI site (25). Recombinant plasmids were subsequently subcloned into the EcoRI-XbaI sites of the expression vector pECE or pcDNA3 (Pharmacia, Piscataway, NJ). Sequence and orientation of the recombinant plasmids were confirmed by dideoxynucleotide analysis (44).

Addition of a NLS [of the SV40 large T antigen, PKKKRKV (45)] between the methionine initiation codon and the first amino acid after the signal peptide (amino acid 20, see Ref 43) of {Delta}-19 mutant long form using the EcoRI and NcoI restriction sites resulted in NLS PRLR mutant (nuclear form); addition of a sequence coding for a Flag epitope in the cDNA of {Delta}-19 and NLS mutants has also been performed for a better visualization of the localization of these receptors (25).

Truncation of the cytoplasmic tail of the Nb2 form resulted in mutant Nb2 T322 (8).

Cotransfection of PRLR cDNA and ß-Casein/Luciferase Reporter Gene in CHO Cells
Biological activities of the different forms and mutants of PRLRs were analyzed using a functional bioassay based on the cotransfection of CHO cells with the expression vector pECE containing cDNAs of the PRLR (and/or mutants PRLR) and a construct that contains a milk protein (ß-casein) gene promoter fused to the reporter gene of the firefly luciferase [plasmid ß-casein/luciferase (46)]. CHO cells were grown on 60-mm culture dishes to 50–60% confluency. Twelve hours before transfection, cells were incubated overnight with GC3 medium (1:1 mixture of DMEM and Ham’s F12 supplemented with transferrin (10 µg/ml), insulin (80 mU/ml), glutamine (2.5 mM), and nonessential amino acids. Cells were transfected by the calcium phosphate procedure (47) with 3 µg long form PRLR/pECE cDNA plus 3 µg DNA carrier (or 6 µg of the mixture of two PRLR cDNAs, e.g. the long and short forms of PRLR/pECE cDNA or two different mutants of PRLR cDNA), 1.5 µg of the fusion gene construct containing the ß-casein/luciferase coding sequence, and 3 µg pCH110 (ß-galactosidase expression vector, Pharmacia, Bromma, Sweden). After incubation for 4 h with the calcium phosphate precipitate, cells were subjected to a 14% (vol/vol) glycerol shock for 2 min and incubated in the presence of oPRL (50 nM; a gift of NIDDK, NIH, Bethesda, MD) and dexamethasone (250 nM, Sigma Chemical Co., St. Louis, MO) or dexamethasone alone for 48 h. Cells were lysed in lysis buffer (Promega, Madison, WI), and centrifuged at 15,000 rpm for 5 min. ß-Casein-luciferase activity was measured in the supernatant as relative light units (Lumat LB 9501, Berthold, Wildbad, Germany); to correct for differences in transfection efficiencies between plates, luciferase activity was normalized to ß-galactosidase activity.

Transient Transfection of 293 Fibroblasts and Luciferase Assay
The human embryonic kidney fibroblast 293 cell line was grown in DMEM/F12 medium containing 10% FCS. Several hours before transfection, cells were plated in a rich medium (2/3 DMEM/F12, 1/3 DMEM containing 4.5 g/liter glucose, and 10% FCS). Then, the cells (5 x 105) were cotransfected with 300 ng of plasmid containing the long and short forms of PRLR cDNA, 500 ng pCH110 (ß-galactosidase), and 100 ng of reporter plasmid that carries the sequence encoding the luciferase gene, which is under the control of a six-repeat sequence of the LHRE DNA, followed by the minimal thymidine kinase promoter (48). Twenty-four hours after transfection, cells were shifted to serum-free medium with or without 18 nM oPRL. After 24 h of stimulation, cells were lysed, and luciferase and ß-galactosidase activation was measured, as described above.

Binding Analysis
COS-7 cells (60–70% confluency) were transiently transfected with the various mutant forms of the PRLR cDNA (5 µg expression plasmid per 100-mm culture dishes), using the diethylaminoethyl/dextran-chloroquine procedure. [125I]hGH was prepared using chloramine T, to a specific activity of 80–140 mCi/mg. The lactogenic hormone human GH binds to the PRLR with the same affinity as oPRL (19). Forty eight hours after transfection, cells were scraped and lysed by three freeze-thaw cycles in 25 mM Tris-HCl, pH 7.4–10 mM MgCl2. After centrifugation at 50,000 x g, 100 µg membranes were incubated with 50,000 cpm [125I]hGH (3 x 108 cpm/µg hGH) in a total volume of 0.4 ml of 25 mM Tris-HCl, pH 7.4–10 mM MgCl2-0.1% BSA. Incubation was carried out at 23 C for 12 h; the microsome suspension was then washed twice with ice-cold Tris-HCl-0.1% PBS and centrifuged at 4000 x g for 30 min to separate bound [125I]hGH from free ligand. Radioactivity of the dried pellet was counted in a {gamma}-counter. Specific binding was defined as the difference between total (in the absence of excess unlabeled ligand) and nonspecific (in the presence of excess ligand, 1 µg/ml) binding. Scatchard analysis was calculated from specific binding obtained in the presence of [125I]hGH with increasing concentrations of unlabeled oPRL (0.1 ng to 10 µg).

Internalization Studies
Experiments were performed according to the procedure established by Vincent et al. (47) as follows. Briefly, COS-7 cells transfected at 70–80% confluency using the diethylaminoethyl/dextran-chloroquine procedure. Forty eight hours after transfection as described above, cells were washed with HEPES binding buffer (24 mM HEPES containing 125 mM NaCl, 4 mM KCl, 1 mM CaCl2, 1.5 mM MgCl2, 2 mM KH2PO4; pH 7.4) and incubated with [125I]hGH (100,000 cpm) in the presence or absence of unlabeled PRL (2.5 µg/well) in 1 ml HEPES binding buffer with 1% BSA for 6 h at 4 C. The kinetics of internalization were determined after different periods (0–60 min) at 37 C. The surface bound [125I]hGH was removed by exposure to an acid wash treatment (NaCl, 0.15 M, glycine, 0.05 M; pH 2.5). Cells were lysed with 1 ml NaOH, 1 M, at room temperature, and lysates’ radioactivity was counted in a {gamma}-counter. Internalization is expressed as the percentage of intracellular [125]IhGH with respect to the total specific cell-associated binding.

Indirect Immunofluorescence
Transfected cells were grown as subconfluent monolayer cultures in Labtek chambers. For the experiments, cells were harvested for 3–12 h in culture medium with 0.1% FBS 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 mouse anti-Flag monoclonal antibody (IBI-A Kodak, New Haven, CT; 10 µg IgG/ml) or mouse monoclonal antibody against the extracellular domain of the PRLR [U5 (49); 60 µg IgG/ml] overnight at 4 C, then washed and further incubated for 45 min with a 1:40 dilution of FITC-conjugated goat anti-IgG, (25). Cells were then mounted in 50% glycerol in PBS and observed on a Zeiss microscope (Carl Zeiss, New York, NY). Specificity control experiments included incubation of cells in the absence of primary and/or secondary antibodies, or with IgG control monoclonal antibodies. No immunofluorescence was detected in any of the specificity control experiments. To confirm the localization of PRLR within the cell, confocal laser scanning microscopy was performed using a Bio-Rad MRC 600 (Bio-Rad Laboratories, Palo Alto, CA), as previously described (50).

Immunoprecipitation
293 cells were stimulated with 18 nM oPRL for 10 min. Then, the cells were lysed in 1 ml of lysis buffer (10 nM 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 dimethylsulfonyl fluoride, 1 µg/ml pepstatin A, 2 µg/ml leupeptin, 5 µg/ml aprotinin) for 10 min at 4 C. Next, the insoluble material was discarded by centrifugation at 12,000 x g for 5 min. Cell lysates were immunoprecipitated overnight at 4 C with 2 µl anti-Flag and 15 µl protein A-sepharose before being washed.

Western Blot Analysis
Proteins were separated on a 7.5% acrylamide gel, transferred on a polyvinylidene difluoride transfer membrane (Polyscreen, Dupont NEN, Boston, MA). Blots were incubated with either an antiphosphotyrosine antibody [4G10, Upstate Biotechnology (Lake Placid, NY) 1 µg/ml], an anti-PRLR monoclonal antibody (U5 at 5 µg/ml) or an anti-Flag antibody (1 µg/ml) for 2 h at room temperature and visualized by enhanced chemiluminescence (ECL) detection (Amersham, Arlington, Heights, IL).


    ACKNOWLEDGMENTS
 
We thank G. Geraud (Institut J. Monod, Paris) for help in confocal laser microscopy and V. Goffin for helpful discussions. Dr. James Ihle kindly provided the Jak2 cDNA and the National Hormone and Pituitary Program, NIDDK, provided the oPRL.


    FOOTNOTES
 
Address requests for reprints to: Dr. Martine Perrot-Applanat, INSERM U 344, Faculté de Médecine Necker, 156 rue de Vaugirard, 75730, Paris Cedex 15, France.

This work was supported by the Institut National de la Santé et de la Recherche Médicale and The Centre National de la Recherche Scientifique.

Received for publication August 7, 1996. Accepted for publication April 8, 1997.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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