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

白细胞介素6 （IL-6）在炎症和癌症中起着关键作用。IL-6受体复合物由IL-6受体（IL-6R）和gp130受体组成。但对IL-6R的胞内结构域（ICD）的作用关注较少，迄今为止，ICD仅被证明需要在极化细胞中对IL-6R进行基底侧分选。到目前为止，还没有发现IL-6R的细胞内结合伙伴。我们采用了两种策略来鉴定IL-6R结合蛋白，第一种是利用IL-6R细胞内结构域的经典酵母-双杂交筛选，第二种是亲和纯化/质谱筛选。酵母-双杂交方法鉴定出MAD2B （REV7）为新的IL-6R结合蛋白。经过数据处理，亲和纯化/质谱筛选，鉴定出4个IL-6R潜在结合蛋白CIP2A、FAM96B、GPN3和SLC25A18。通过共沉淀实验独立验证了IL-6R与MAD2B的相互作用。发现IL-6R中的MAD2B结合基序与IL-6R的主要基底侧排序基序重叠。因此，我们假设MAD2B可能参与了IL-6R的基底外侧分选。此外，我们将分析il -6诱导的信号转导在IL-6R及其新型相互作用蛋白的胞内结构域的动力学。MAD2B是一种泛素连接酶CDH1-APC和CDC20-APC的抑制剂，可以假设MAD2B也可能具有抑制其他泛素连接酶的作用，如c-Cbl。C-Cbl负责gp130泛素化、内化、降解和信号终止。CIP2A（癌性PP2A抑制剂）是蛋白磷酸酶2A （PP2A）的抑制剂。抑制PP2A增加gp130的ser782磷酸化，这是gp130的内化和蛋白酶体降解以及随后抑制il -6诱导的信号转导的原因。我们很容易推测，与IL-6R结合的CIP2A可能会阻断局部PP2A，从而增加gp130的内在化，从而抑制信号传导的诱导。最后，我们将研究IL-6R在小鼠细胞内结构域的作用。为此，我们将在缺乏IL-6R细胞内结构域的小鼠中产生新的IL-6R敲除。我们将分析IL-6R基底侧分选的破坏和/或MAD2B和/或其他结合蛋白与IL-6R的相互作用是否会调节体内的信号转导，这可能对IL-6介导的病理生理如炎症和癌症有直接影响。

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.