Liganden-unabhängige Aktivierung gp130-ähnlicher Rezeptoren durch erzwungene Homo- und Heterodimerisierung

The IL-6 family of cytokines consists of IL-6, vIL-6, IL-11, IL-27, CNTF, CT-1, LIF, CLC, NP, OSM, and IL-31. IL-6, vIL-6, IL-11 induce the formation of a gp130-homodimer, whereas signaling by CNTF, CT-1, LIF, CLC, and NP results in the formation of a gp130/LIFR heterodimer. OSM can induce the formation of a dimer of gp130 with LIFR or the related protein OSMR. IL-27 exclusively signals via a heterodimer comprising gp130 and WSX-1, whereas IL-31 induces the formation of an OSMR/GPL heterodimer. Interestingly, naturally occurring, ligand-independent, and constitutively active gp130 variants were described to play a role in the onset of inflammatory hepatocellular adenomas. In the past, constitutively active gp130 variants based on homodimerization of Jun leucine zippers were generated in this laboratory. However, using a Fos leucine zipper-gp130 (Fos-gp130) chimera and a Jun leucine zipper-WSX-1 chimera (Jun-WSX-1), Fos and Jun form the AP-1 transcription factor complex, revealed that also Fos-gp130 alone was able to induce ligand-independent STAT3 phosphorylation and ligand-independent growth of cytokine-dependent cells indicating that this approach was not suitable for the generation of ligand-independent, constitutive activation of heterodimeric receptors. Therefore a novel IL-15/IL-15Rα based system to generate ligand-independent, constitutively active heterodimeric complexes for all known gp130-receptor members was developed. In this system, IL-15 was genetically fused to one receptor component whereas IL-15Rα was fused to the second receptor component. Ligand-independent heterodimerization of gp130 with WSX-1, LIFR and OSMR and of OSMR with GPL led to constitutive, ligand-independent STAT1 and/or STAT3 and ERK1/2 phosphorylation. Moreover, these receptor combinations induced transcription of the STAT3 target genes c-myc and Pim-1 upon stable transfection and conferred to independent growth of cytokine-dependent cells. The IL-15/IL-15Rα system is established as a novel system to mimic constitutive and ligand-independent activation of homo- and heterodimeric receptor complexes, which could also be applicable to other heterodimeric cytokine receptor and growth factor receptor families. Additionally, in an improved approach a mutated IL-15 protein was used, which was still able to bind the IL-15R-sushi domain, but not to IL-2Rβ and IL-2Rγc receptor chains. In combination with the 2A peptide technology, this is the first step towards to translation of the in vitro data to analyse the tumorigenic potential of gp130-heterodimeric receptor complexes in vivo.Die IL-6 Zytokinfamilie besteht aus IL-6, vIL-6, IL-11, IL-27, CNTF, CT-1, LIF, CLC, NP, OSM und IL-31. IL-6, vIL-6 und IL-11 induzieren die Bildung eines gp130 Homodimers, wohingegen die Signalwirkung von CNTF, CT-1, LIF, CLC und NP zur Bildung eines gp130/LIFR Heterodimers führen. OSM induziert die Bildung eines Dimers aus gp130 und LIFR oder dem verwandten Protein OSMR. IL-27 betreibt ausschließlich Signalwirkung durch ein Heterodimer bestehend aus gp130 und WSX-1, wohingegen IL-31 die Bildung eines OSMR/GPL Heterodimers induziert. Interessanterweise wurden natürlich vorkommende, Liganden-unabhängige und konstitutiv aktive gp130 Varianten beschrieben, welche eine Rolle bei der Entstehung von entzündlichen hepatozellulären Adenomen spielen. In der Vergangenheit wurde in dieser Arbeitsgruppe eine Liganden-unabhängige und konstitutiv aktive gp130 Variante, auf der Homodimerisierung von Jun „Leucinzippern“ basierend, hergestellt. Allerdings, unter Verwendung einer Fos „Leucinzipper“ gp130 Chimäre (Fos-gp130) und einer Jun „Leucinzipper“ WSX-1 Chimäre (Jun-WSX-1), Fos und Jun bilden den AP-1 Transkriptionsfaktorkomplex, stellte sich heraus, dass Fos-gp130 bereits alleine in der Lage ist eine Liganden-unabhängige STAT3-Phosphorylierung und unabhängiges Wachstum von Zytokin-abhängigen Zellen zu induzieren. Dieser Ansatz konnte daher nicht für die Herstellung von Liganden-unabhängigen, konstitutiv aktiven heterodimeren Rezeptorkomplexen verwendet werden. Daher wurde ein neues auf IL-15/IL-15Rα-sushi basierendes System zur Herstellung von Liganden-unabhängigen und konstitutiv aktiven Heterodimeren aller gp130 ähnlichen Rezeptoren entwickelt. In diesem System wurde eine Rezeptorkomponente mit IL-15, die andere mit der sushi Domäne des IL-15Rα, fusioniert. Liganden-unabhängige Heterodimerisierung von gp130 mit WSX-1, LIFR und OSMR bzw. OSMR mit GPL führte zur konstitutiven, liganden-unabhängigen STAT1- und/oder STAT3- und ERK1/2-Phosphorylierung. Darüber hinaus führten diese Rezeptorkombinationen zur Induktion der Transkription der STAT3 Zielgene c-myc und Pim-1 und zu unabhängigem Wachstum von Zytokin-abhängigen Zellen. Die Etablierung des neuartigen IL-15/IL-15Rα-sushi basierenden Systems, zur Nachahmung von konstitutiver, Liganden-unabhängiger Aktiverung homo- oder heterodimerer Rezeptorkomplexe, kann wohlmöglich auch auf andere heterodimere Zytokinrezeptor- oder Wachstumsfaktorrezeptor-Familien anwendbar sein. Zusätzlich wurde in einem verbesserten Ansatz ein mutiertes IL-15 Protein verwendet, welches zwar noch an die IL-15Rα-sushi Domäne, allerdings nicht mehr an die IL-2Rβ und IL-2Rγc Rezeptoren, binden kann. Kombiniert mit der 2A Peptid Technology, ist dies der erste Schritt die in vitro Daten anzuwenden, um das tumorigene Potential von gp130 heterodimeren Rezeptorkomplexen in vivo zu analysieren

Unraveling Viral Interleukin-6 Binding to gp130 and Activation of STAT-Signaling Pathways Independently of the Interleukin-6 Receptor▿ ‡

Human herpesvirus 8 encodes a viral version of interleukin-6 (vIL-6) which shows 25% sequence homology with human IL-6. In contrast to human IL-6, which first binds to the IL-6 receptor (IL-6R) and only subsequently associates with the signal transducing receptor subunit gp130, vIL-6 has been shown to directly bind to gp130 without the need of IL-6R. As a functional consequence, vIL-6 can activate far more target cells in the body since all cells express gp130, but only cells such as hepatocytes and some leukocytes express IL-6R. We sought to understand which amino acid sequences within the vIL-6 protein were responsible for its ability to bind and activate gp130 independent of IL-6R. As a first approach, we constructed chimeric IL-6 proteins in which all known gp130 interacting sites (sites II and III) were sequentially transferred from vIL-6 into the human IL-6 protein. To our surprise, human IL-6 carrying all gp130 interacting sites from vIL-6 did not show IL-6R-independent gp130 activation. Even more surprisingly, the loop between helix B and C of vIL-6, clearly shown in the crystal structure not to be in contact with gp130, is indispensable for direct binding to and activation of gp130. This points to an IL-6R induced change of site III conformation in human IL-6, which is already preformed in vIL-6. These data indicate a novel activation mechanism of human IL-6 by the IL-6R that will be important for the construction of novel hyperactive cytokine variants

N-Linked Glycosylation Is Essential for the Stability but Not the Signaling Function of the Interleukin-6 Signal Transducer Glycoprotein 130*

N-Linked glycosylation is an important determinant of protein structure and function. The interleukin-6 signal transducer glycoprotein 130 (gp130) is a common co-receptor for cytokines of the interleukin (IL)-6 family and is N-glycosylated at 9 of 11 potential sites. Whereas N-glycosylation of the extracellular domains D1–D3 of gp130 has been shown to be dispensable for binding of the gp130 ligand IL-6 and its cognate receptor in vitro, the role of the N-linked glycans on domains D4 and D6 is still unclear. We have mutated the asparagines of all nine functional N-glycosylation sites of gp130 to glutamine and systematically analyzed the consequences of deleted N-glycosylation (dNG) in both cellular gp130 and in a soluble gp130-IgG1-Fc fusion protein (sgp130Fc). Our results show that sgp130Fc-dNG is inherently unstable and degrades rapidly under conditions that do not harm wild-type sgp130Fc. Consistently, the bulk of cellular gp130-dNG is not transported to the plasma membrane but is degraded in the proteasome. However, the small quantities of gp130-dNG, which do reach the cell surface, are still able to activate the key gp130 signaling target signal transducer and activator of transcription-3 (STAT3) upon binding of the agonistic complex of IL-6 and soluble IL-6 receptor. In conclusion, N-linked glycosylation is required for the stability but not the signal-transducing function of gp130

Forced Homo- and Heterodimerization of All gp130-Type Receptor Complexes Leads to Constitutive Ligand-independent Signaling and Cytokine-independent Growth

We present a novel strategy to enforce cytokine-independent, constitutive signaling of heterodimeric gp130 receptor complexes. Replacing the extracellular domain of gp130-type receptors by IL-15/IL-15R is sufficient to heterodimerize gp130-like receptors and as a consequence leading to sustained cytokine-independent receptor activation