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-6 R）和gp 130受体组成。只有很少的注意到的IL-6 R的细胞内结构域（ICD）的作用，这是迄今为止只显示在极化细胞中的IL-6 R的基底外侧分选所需的。迄今为止，没有描述IL-6 R的细胞内结合配偶体。我们应用两种策略来鉴定IL-6 R结合蛋白，首先是使用IL-6 R的胞内结构域的经典酵母双杂交筛选，其次是亲和纯化/质谱筛选。酵母双杂交方法导致MAD 2B（REV 7）被鉴定为新的IL-6 R结合蛋白。数据处理后，亲和纯化/质谱筛选导致鉴定出四种潜在的IL-6 R结合蛋白CIP 2A、FAM 96 B、GPN 3和SLC 25 A18。通过共沉淀实验独立地验证IL-6 R与MAD 2B的相互作用。发现IL-6 R中的MAD 2B结合基序与IL-6 R的优势基底外侧分选基序重叠。因此，我们假设MAD 2B可能参与IL-6 R的基底外侧分选。此外，我们将分析IL-6诱导的信号转导与IL-6 R的细胞内结构域及其新的相互作用蛋白的动力学。MAD 2B是遍在蛋白连接酶CDH 1-APC和CDC 20-APC的抑制剂，并且可以假设MAD 2B也可能在抑制其他遍在蛋白连接酶（例如c-Cabl）中发挥作用。C-Cbl负责gp 130的泛素化、内化、降解和信号终止。CIP 2A（PP 2A的癌性抑制剂）是蛋白磷酸酶2A（PP 2A）的抑制剂。PP 2A的抑制增加了gp 130的Ser 782-磷酸化，这是负责gp 130的内化和蛋白酶体降解以及随后的IL-6诱导的信号转导的抑制。推测与IL-6 R结合的CIP 2A可能阻断局部PP 2A，从而增加gp 130内化以抑制信号传导的诱导是诱人的。最后，我们将研究IL-6 R的细胞内结构域在小鼠中的作用。为此，我们将在缺乏IL-6 R胞内结构域的小鼠中产生新的IL-6 R敲除。我们将分析IL-6 R的基底外侧分选的破坏和/或MAD 2B和/或另外的结合蛋白与IL-6 R的相互作用是否调节体内信号转导，这可能对IL-6介导的病理生理学如炎症和癌症具有直接后果。

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.