We have investigated mechanisms and consequences of STAT5 activation through the human IL-4 receptor (IL-4R). By functionally expressing receptor mutants in the murine pro-B cell line Ba/F3, we could show that phosphorylated tyrosine residues within the IL-4R alpha chain are dispensable for IL-4-induced STAT5 activity. However, disruption of a membrane-proximal proline-rich sequence motif ('box1') in either subunit of the bipartite IL-4R abolished not only ligand-induced tyrosine phosphorylation of Janus kinases JAK1 and JAK3, but also IL-4-triggered activation of STAT5 and concomitant cell proliferation. A dominant-negative version of STAT5b, but not of STAT5a, interfered with IL-4-induced DNA synthesis in Ba/F3 cells, suggesting an involvement of STAT5b in the control of cell proliferation through IL-4R. Reporter gene experiments finally showed that transcription from promoters of STAT5 target genes can be specifically induced by challenging cells with IL-4, and that both STAT5a and STAT5b can contribute to IL-4-triggered transcriptional control.

Interleukin-4 (IL-4) triggers cellular responses by interaction with the bipartite interleukin-4 receptor (IL-4R). IL-4-responsive cells specifically endocytose IL-4. We studied the ligand internalization properties of the human IL-4R and analyzed the specific functions of its two subunits IL-4Ra and gc in this process. IL-4 mutant RY, which binds to IL-4Ra but does not recruit gc into the receptor complex was used as a tool to show that IL-4Ra can promote independent ligand uptake in human T cells. Internalization was limited, however, by rapid IL-4 dissociation, suggesting that one important function of gc in IL-4 endocytosis is to retain the ligand sufficiently long within the ternary receptor complex. We then measured IL-4 internalization by murine Ba/F3 cells that were stably transfected with various human IL-4R constructs. Efficient IL-4 uptake required the cytoplasmic section of the receptor. The intracellular domains of IL-4Ra and gc were responsible for independent endocytosis processes with distinct kinetics. IL-4Ra-mediated internalization resulted in long-term intracellular maintainance of IL-4, whereas gc directed the associated radioligand to intracellular breakdown and rapid release in the form of degraded protein. Mutants of either IL-4R subunit deficient in Janus kinase activation were not impaired in internalization, indicating that IL-4 endocytosis is not functionally connected to signal transduction.Keywords: cytokine receptor; endocytosis; internalization; interleukin-4; signal transduction.Interleukin-4 (IL-4) is a key regulator of the immune system and is a central mediator of type I allergy [1]. It exerts its function on various target cells by activating the bipartite interleukin-4 receptor (IL-4R), which consists of the IL-4Ra subunit and the common g receptor chain (gc). The latter is also a constituent of the receptor systems for IL-2, IL-7, IL-9 and IL-15 [2]. The interaction of IL-4 with the IL-4R in the course of heterodimerization is a crucial event for the onset of IL-4 driven cellular reactions and has therefore been extensively studied, also with the aim of potential pharmacological intervention. Antagonistic inhibitors of IL-4 have been developed based on their properties to tightly bind IL-4Ra, but to fail contacting gc [3].Ligand-induced dimerization of IL-4Ra and gc triggers signal cascades that involve reversible tyrosine phosphorylation of cytoplasmic proteins, e.g. the JAK/STAT pathway [4]. Whereas the initiation of these signal transduction events has been quite well characterized in recent years, the limitations and termination of IL-4-induced signaling is still poorly understood. One important general mechanism of signal down-regulation and control of cell responsiveness is the internalization of ligand±receptor complexes. Endocytosis of IL-4 by IL-4R-expressing cells has been demonstrated [5], but has not yet been investigated in detail.The most thoroughly analyzed endocytosis pathway proceeds via clathrin-coated pits as examplified by the transferrin receptor [6]. T...

Stimulation of susceptible cells by interleukin-4 leads to activation of signal transducer and activator of transcription (STAT6) through tyrosine phosphorylation and dimerisation, thus directing it to the cell nucleus and rendering it a sequence-specific transcription factor. We functionally reconstituted human interleukin-4 receptor complexes with intracellular truncations of either the A or γ subunits and demonstrate the requirement for elements from both receptor chains for STAT6 activation induced by interleukin-4. By assaying the signalling properties of human interleukin-4-receptor A-chain-deletion constructs in both Ba/F3 cells and COS-7 cells, we show that all its cytoplasmic tyrosine residues can be removed without affecting the capability of the receptor complex to trigger STAT6 function with regard to tyrosine phosphorylation, DNA binding, and specific gene transcription. The activation of both STAT6 and janus kinase 1 (JAK1) by the interleukin-4 receptor was completely abolished by disruption of the membraneproximal 'box1' motif in the interleukin-4 receptor A chain. Our results indicate a redundant role of the previously defined phosphotyrosine-containing STAT6 docking site and suggest a mechanism of immediate activation of STAT6 by receptor-associated janus kinase(s). In addition, we demonstrate that dominant negative versions of both interleukin-4 receptor subunits are able to block interleukin-4 induced signalling via STAT6.Keywords : signal transduction; cytokine receptor ; transcription factor; tyrosine phosphorylation; Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway.A considerable variety of mammalian cell types is able to receptor stimulation was shown to be the phosphorylation and respond to the immunoregulatory cytokine interleukin-4 (IL-4) activation of STAT6. STAT6 is a member of the family of signal [1]. All these target cells express a functional IL-4 receptor transducer and activator of transcription proteins, which in re-(IL-4R), which comprises the ligand-binding IL-4RA subunit [2] sponse to receptor-mediated activation dimerise and translocate and, in lymphocytes, the common γ receptor chain (γc) as signal-to the nucleus where they interact with cognate sequences in the ling mediator [3,4]. Both receptor proteins belong to the hemo-DNA and modulate the transcription of specific genes [11]. The poietin-receptor superfamily [5]. Upon stimulation by interac-JAK-STAT signalling pathway, first discovered in the interferon tion with IL-4, a dimerisation of IL-4 receptor subunits occurs receptor system [12Ϫ14], is now generally regarded as a paraand triggers a program of cytoplasmic reactions that ultimately digm of signal transduction by cytokine receptors, which by lead to specific gene regulation and long-term cellular processes themselves do not comprise an intrinsic tyrosine kinase activity such as proliferation and differentiation [6].[15]. Besides tyrosine phosphorylation of the receptor itself, of the STAT6 was originally characterised as a mediato...

The bipartite human interleukin-4 (IL-4) receptor was functionally expressed in murine pro-B cells and activated by human IL-4 to evoke intracellular signaling. Mutual association of signal transducing proteins within the receptor complex was then studied in dependence of ligand stimulation. Besides ligandinduced receptor heterodimerization and contacts of the two IL-4 receptor subunits K K and Q Q with Janus kinases JAK1 and JAK3 a prominent constitutive binding between JAK1 and signal transducer and activator of transcription STAT5 was detected. Since both these proteins become phosphorylated in response to IL-4 receptor stimulation, the influence of tyrosine phosphorylation on their mutual contact was analyzed. Association of JAK1 and STAT5 was found to occur exclusively between unphosphorylated proteins.z 1998 Federation of European Biochemical Societies.