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

白细胞介素 6 受体信号传导中的膜分选和膜相关蛋白复合物

• 批准号: 232771704
• 负责人: Professor Dr. Jürgen Scheller
• 金额: --
• 依托单位: Institut für Biochemie und Molekularbiologie II
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/232771704?language=en
• 关键词: Membrane; sorting; membrane; associated; protein

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.

白细胞介素 6 (IL-6) 在炎症和癌症中发挥着关键作用。 IL-6受体复合物由IL-6受体(IL-6R)和gp130受体组成。很少有人关注 IL-6R 胞内结构域 (ICD) 的作用，迄今为止仅表明它是极化细胞中 IL-6R 基底外侧分选所必需的。迄今为止，尚未描述 IL-6R 的细胞内结合配偶体。我们应用两种策略来鉴定 IL-6R 结合蛋白，首先是使用 IL-6R 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 MAD2B (REV7) 被鉴定为新型 IL-6R 结合蛋白。数据处理后，亲和纯化/质谱筛选鉴定出四种潜在的 IL-6R 结合蛋白 CIP2A、FAM96B、GPN3 和 SLC25A18。 IL-6R 与 MAD2B 的相互作用通过共沉淀实验独立验证。发现 IL-6R 中的 MAD2B 结合基序与 IL-6R 的主要基底外侧分选基序重叠。因此，我们假设 MAD2B 可能参与 IL-6R 的基底外侧分选。此外，我们将分析 IL-6 诱导的信号转导相对于 IL-6R 胞内结构域及其新型相互作用蛋白的动态。 MAD2B 是泛素连接酶 CDH1-APC 和 CDC20-APC 的抑制剂，并且可以假设 MAD2B 也可能在抑制其他泛素连接酶（例如 c-Cbl）中发挥作用。 C-Cbl 负责 gp130 泛素化、内化、降解和信号终止。 CIP2A（PP2A 癌抑制剂）是蛋白磷酸酶 2A (PP2A) 的抑制剂。抑制 PP2A 会增加 gp130 的 Ser782 磷酸化，从而导致 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。人们很容易推测与 IL-6R 结合的 CIP2A 可能会阻断局部 PP2A，从而增加 gp130 内化以抑制信号传导的诱导。最后，我们将研究 IL-6R 胞内结构域在小鼠中的作用。为此，我们将在缺乏 IL-6R 胞内结构域的小鼠中产生新型 IL-6R 敲除。我们将分析，IL-6R 基底外侧分选的破坏和/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信号转导，这可能对 IL-6 介导的病理生理学（例如炎症和癌症）产生直接影响。