N-glycosylation of the prolactin receptor is not required for activation of gene transcription but is crucial for its cell surface targeting.

The functional importance of the three oligosaccharide chains linked to Asn35, Asn80 and Asn108, of the long form of the PRL receptor (PRLR) was investigated by individual or multiple substitutions of asparagyl residues using site-directed mutagenesis and transient transfection of these mutated forms of PRLR in monkey kidney cells, Chinese hamster ovary, and human 293 fibroblast cells that exhibit different levels of protein expression. Scatchard analysis performed on monkey kidney cells revealed that the mutants possess the same affinity for PRL as compared with wild-type PRLR. A strong reduction (90%) of the aglycosylated PRLR expression at the cell surface of monkey kidney or human 293 cells was observed. Immunohistochemistry experiments using an anti-PRLR monoclonal antibody showed an accumulation of the deglycosylated receptor in the Golgi area of transfected monkey kidney cells. Upon PRL stimulation, the aglycosylated PRLR associated with Janus kinase 2 was phosphorylated and was able to activate a beta-casein gene promoter in transfected 293 fibroblast cells. The active form of the PRLR was thus acquired independently of glycosylation. By contrast, no functional activity was detectable in transfected Chinese hamster ovary cells that expressed low levels of PRLR. These studies demonstrate that the glycosylation on the asparagyl residues of the extracellular domain of the PRLR is crucial for its cell surface localization and may affect signal transduction, depending on the cell line.

Interactions among Janus kinases and the prolactin (PRL) receptor in the regulation of a PRL response element.

PRL regulates milk gene expression, at least in part, by activating JAK2 kinase and STAT5 (signal transducer and activator of transcription 5), initially termed mammary gland factor (MGF). These experiments were initiated to gain a better understanding of the mechanisms of transcriptional activation via PRL receptor (PRL-R) signaling. Binding of PRL to the recombinant pigeon PRL-R-activated transcription driven by a 2.8 kbp 5'-fragment of the rat beta-casein gene. PRL enhanced the expression of chimeric reporters containing the beta-casein PRL response element (PRE), but not the c-fos sis-inducible element, when the reporters were transfected into Chinese hamster ovary cells with the PRL-R. Wild type receptor, which contains a duplication of the entire extracellular ligand-binding domain, was only slightly more effective than a truncation mutant with a single extracellular domain. Transfection with either JAK1, JAK2, or JAK3 increased basal transcription through both the PRE and sis-inducible element. Coexpression of JAK2 with PRL-R resulted in amplification of the induction of the PRE by PRL, whereas JAKs 1 and 3 did not amplify the PRL effect. Overexpression of JAK2 mutants blocked PRE activation by PRL. Mutant JAK2 also interfered with PRE activation by JAK3 but did not affect JAK1's stimulatory effect.

Homodimerization of IL-2 receptor beta chain is necessary and sufficient to activate Jak2 and downstream signaling pathways.

Cytokine receptor signaling involves the Jak/Stat pathways. Heterotrimeric IL-2R (alpha, beta, gamma[c] chains) activates Jak1 and Jak3, whereas homodimeric PRLR activates Jak2. The requirements directing such specificity of Jak activation are unknown. We show that chimeric receptors containing the intracellular domain of IL-2Rbeta chain fused to the extracellular domain of either EPOR or Kit, a non-cytokine receptor, activate Jak2. This observation provides evidence that IL-2Rbeta intrinsically possesses the ability to activate Jak2, but that this property is only displayed in homodimerized complexes. Our data suggest a role for the stoichiometry of cytokine receptors in selective activation of Janus kinases.

The last proline of Box 1 is essential for association with JAK2 and functional activation of the prolactin receptor.

The interaction of prolactin (PRL) with its receptor leads to activation of the tyrosine kinase, Janus kinase 2 (JAK2). In the cytoplasmic juxtamembrane region, a short segment (Box 1) which is conserved in other receptors of the PRL/growth hormone (GH)/cytokine receptor family, is required for signal transduction. To assess the contribution of the different amino acids of Box 1, individual alanine substitutions of all residues, grouped substitution of four prolines (4PA mutant) and individual leucine replacement of the two last prolines (P248L and P250L mutants) were introduced. Here we show that P250L and 4PA (i) inhibit PRL-induced transactivation of a luciferase reporter governed by a beta-caseine gene promoter; (ii) decrease in JAK2 tyrosine kinase activity in biotinylated-PRL precipitates; (iii) impair the interaction between PRLR and JAK2, as evidenced by lack of co-immunoprecipitation, (iv) and prevent the activation of signal transducer and activator of transcription (Stat) as determined by absence of tyrosine phosphorylation of Stat5. Our data suggest that the Box 1 region of the PRL receptor and particularly the last proline is critical for JAK2 association and subsequent activation. These results support the notion that the tyrosine kinase JAK2 is implicated in activation of downstream protein effectors such as Stat5, which are involved in transcription of PRL-responsive genes.

Functional activity of the human prolactin receptor and its ligands.

Prolactin receptors (PRLR) have been identified in a number of human tissues and cell lines, although little is known about the human receptor protein. The cloning of the human PRLR cDNA has enabled further characterization of the receptor protein in transfected cells. Since the human cDNA is expressed at lower levels than the rat cDNA, we have constructed a hybrid cDNA (pECE r5'hPRLR) containing nucleotides of the 5' untranslated region and signal peptide of the rat PRLR and the protein coding and 3' untranslated portion of the human receptor. Expression of the hybrid receptor was increased more than two-fold compared to the human receptor as detected by specific binding of 125I-human growth hormone (GH) to transfected COS-7 cells. The relative molecular mass of the receptor was 93,000 Da, as determined by chemical cross-linking studies. Transcriptional assays were used to show the human PRLR was able to activate two milk protein genes; ovine beta-lactoglobulin and rat beta-casein. Transfected cells expressing the human PRLR receptor, treated with human GH or prolactin (PRL), induced a dose-dependent increase in transcriptional activation of the beta-casein/luciferase fusion gene. Glycosylated, and non-glycosylated human PRL, and ovine PRL were equally effective in activating the beta-casein promoter. Human placental lactogen and bovine PRL could also induce a greater than 10-fold induction, whereas insulin did not significantly stimulate the beta-casein promoter. The results show that the human PRLR can activate both beta-lactoglobulin and beta-casein milk gene promoters and that these reporter genes can be used to evaluate the functional activity of agonists and antagonists of the human PRLR.

Immune function of prolactin (PRL) and signal transduction by PRL/GH/cytokine receptors: specificity, redundancy and lessons from chimaeras.

Although prolactin (PRL) was originally regarded exclusively as a lactogenic hormone, there are a number of observations that suggest a role for this protein in the regulation of immune responses. The first step in understanding this unexpected function came from the cloning of the prolactin receptor, which was later shown to be a member of the cytokine receptor superfamily. The PRL receptor shares structural analogies with receptors for proteins acting on immune cells, the prototype of which is IL-2. Studies of cytokine receptor signalling revealed that all messages are transmitted in the cell through a limited set of transducers, among which the JAK kinases and the Stat transcription factors represent a major cascade. Deciphering the rules allowing a given cytokine receptor, and not another, to activate a particular set of JAK and Stat proteins is a key step in understanding functional specificities within this receptor superfamily. Mutational analyses have provided interesting information about which features are required for which property. Much less data are available from studies using chimaeric receptors, although this strategy is probably more powerful for comparing different receptors and addressing the question of their specificity (or redundancy). As frequently as possible, we shall illustrate our discussions through experimental investigations using the chimaeric approach.

Prolactin activates tyrosyl phosphorylation of insulin receptor substrate 1 and phosphatidylinositol-3-OH kinase.

Prolactin (PRL) has been demonstrated to induce tyrosine phosphorylation and activation of the cytoplasmic tyrosine kinase JAK2. The present study represents an initial effort to identify the phosphorylation repertoire of the PRL receptor (PRLR). For this purpose we have modified the rat PRLR cDNA to encode an additional N-terminal epitope specifically designed to allow the rapid purification of the PRLR and associated proteins from transfected cells. The Flag-tagged PRLR was stably expressed in the human 293 cell line. PRL induced tyrosine phosphorylation of proteins of 85, 95, and 185 kDa from 10 to 30 min after PRL stimulation. Immunoblot analysis of immunoprecipitation indicates that p85 corresponds to the 85-kDa regulatory subunit of phosphatidylinositol (PI)-3' kinase, p95 to PRLR, and p185 to insulin receptor substrate 1 (IRS-1). Both PI-3' kinase and IRS-1 appear to associate with PRLR in a PRL-dependent manner. These results thus indicate that kinases other than JAK2, namely PI-3' kinase, are activated by PRL.

Internalization of prolactin receptor and prolactin in transfected cells does not involve nuclear translocation.

Prolactin (PRL) interacts with a specific, well characterized plasma membrane receptor (PRLR) that is coupled to signal transduction pathways involving Jak2, Fyn, and MAP kinases, and signal transducers and activators of transcription (STAT). Although a few previous studies have indicated nuclear translocation of PRL in IL-2 stimulated T lymphocytes, PRL-dependent Nb2 lymphoma cell lines and 235-1 lactotrophs, the mechanisms of nuclear targeting remain unknown and conflicting results have been reported concerning the putative nuclear translocation of the PRLR. We therefore decided to investigate nuclear translocation of PRLR and PRL in various cell lines transfected with an expression plasmid encoding PRLR, using confocal laser microscopy. We have constructed various cDNAs of the long and short forms of the rat PRLR containing an oligonucleotide encoding a Flag epitope inserted either just before the N-terminal amino acid or in the C-terminal end of the mature receptor (named N-terminal or C-terminal Flag-tagged PRLR). The corresponding receptors function as the PRLR in transfected cells: they are expressed at the plasma membrane and in compartments of the secretory pathway, they bind PRL with normal affinity (Kd= 4x10(-10) M) and have the same capacity to stimulate the transcriptional activity of a milk protein (beta-casein) gene as wild-type PRLR. In addition, the tagged receptors are much more efficiently immunodetected using anti-Flag antibodies, as compared to anti-PRL antibodies (U5 or U6). Immunofluorescence combined with detailed confocal laser microscopy showed that addition of PRL (0 to 12 hours) to COS-7, CHO and NIH-3T3 transfected fibroblasts induces rapid internalization of the receptor (long form), without any translocation to the nucleus. Using PRL-R tagged both in the N-terminal or C-terminal regions of the mature receptor excludes the possibility of a cleaved fragment which could have been subsequently imported into the nucleus. An absence of nuclear translocation of PRLR was also observed in a 293 cell line stably expressing the receptor, and in physiological targets for PRL, i.e. in Nb2 lymphoma cells expressing the Nb2 form of the receptor or in BGME mammary gland epithelial cells upon overexpression of a Flag-tagged PRLR. Similarly, the short form of the PRLR was not detected in nuclei of transfected COS cells upon PRL treatment. Clearly, our results provide evidence that internalization of the plasma membrane PRLR does not lead to nuclear translocation of the receptor, or part of it, in most fibroblasts and epithelial cells at physiological concentrations of PRL. Also, in co-localization experiments, PRL was internalized without nuclear translocation. Activation of STATs transcription factors and MAP kinases, as well as translocation of these proteins to the nucleus following their phosphorylation, probably remains the intracellular mechanism coupling stimulation to nuclear events.

Null mutation of the prolactin receptor gene produces multiple reproductive defects in the mouse.

Mice carrying a germ-line null mutation of the prolactin receptor gene have been produced by gene targeting in embryonic stem cells. Heterozygous females showed almost complete failure of lactation attributable to greatly reduced mammary gland development after their first, but not subsequent, pregnancies. Homozygous females were sterile owing to a complete failure of embryonic implantation. Moreover, they presented multiple reproductive abnormalities, including irregular cycles, reduced fertilization rates, defective preimplantation embryonic development, and lack of pseudopregnancy. Half of the homozygous males were infertile or showed reduced fertility. This work establishes the prolactin receptor as a key regulator of mammalian reproduction, and provides the first total ablation model to further study the role of the prolactin receptor and its ligands.