Membrane sorting and membrane-associated protein complexes in interleukin 6 receptor signaling

• Applicant: Professor Dr. Jürgen Scheller
• Subject Area: Cell Biology
• Term: from 2013 to 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 232771704

Interleukin 6 (IL-6) plays a critical role in inflammation and cancer. The IL-6 receptor complex consists of the IL-6 receptor (IL-6R) and the gp130 receptor. Only little attention was paid to the role of the intracellular domain (ICD) of the IL-6R, which is so far only shown to be required for basolateral sorting of IL-6R in polarized cells. No intracellular binding partner of the IL-6R was described to date. We applied two strategies to identify IL-6R binding proteins, firstly a classical yeast-two-hybrid screen using the intracellular domain of the IL-6R and secondly an affinity purification/mass spectrometry screen. The yeast-two-hybrid approach led to the identification of MAD2B (REV7) as novel IL-6R binding protein. After data processing, the affinity purification/mass spectrometry screen led to the identification of four potential IL-6R binding proteins CIP2A, FAM96B, GPN3 and SLC25A18. The interaction of IL-6R with MAD2B was verified independently by co-precipitation experiments. The MAD2B binding motif in the IL-6R was found to overlap with the dominant basolateral sorting motif of the IL-6R. Accordingly, we hypothesize that MAD2B might be involved in basolateral sorting of the IL-6R. Furthermore, we will analyze the dynamics of IL-6-induced signal transduction with respect to the intracellular domain of the IL-6R and its novel interacting protein. MAD2B is an inhibitor of the ubiquitin-ligases CDH1-APC and CDC20-APC and it can be assumed that MAD2B might also have a role in inhibition of other ubiquitin-ligases, such as c-Cbl. C-Cbl is responsible for gp130 ubiquitination, internalization, degradation and signal termination. CIP2A (cancerous inhibitor of PP2A) is an inhibitor of protein phosphatase 2A (PP2A). Inhibition of PP2A increases Ser782-phosphorylation of gp130, which is responsible for internalization and proteasomal degradation of gp130 and subsequent inhibition of IL-6-induced signal transduction. It is tempting to speculate that CIP2A bound to IL-6R might block local PP2A, thereby increases gp130 internalization to dampen induction of signaling. Finally, we will study the role of the intracellular domain of the IL-6R in mice. To this end, we will generate novel IL-6R knock in mice lacking the intracellular domain of IL-6R. We will analyze, if disruption of basolateral sorting of IL-6R and/or interaction of MAD2B and/or additional binding proteins with IL-6R modulate signal transduction in vivo, which might have direct consequences for IL-6 mediated pathophysiology such as inflammation and cancer.

• DFG Programme: Research Grants