Targeted Engineering of Designer Arrestins to Regulate Cell Signaling

项目总结 阻滞素被发现是G蛋白偶联受体(GPCR)信号的负性调节因子。 蛋白质。新数据显示，游离和受体结合的拦阻蛋白通过MAP启动信号传递 激酶，它调节细胞的死亡、存活和增殖。例如，游离的和受体结合的 Arrestin-3支架通过ASK1-MKK4/7-JNK1/2/3级联，促进JNK家族的激活。 这两种非视觉阻滞剂与人体内约800个不同的GPCR相互作用。我们建议确定 Arrestin元件负责受体的特异性，并阐明组装的结构基础 由拦阻蛋白组织的多蛋白信号复合体(信号体)。我们的新晶体结构 Arrestin-3在丰富的细胞质分子IP6存在下显示受体结合样(活性) Arrestin-3的构象。Arrestin-3在缺乏GPCRs的情况下呈现这种构象的能力 可能解释了arrestin-3的受体非依赖性支架活性。我们鉴定了arrestin-3元素 对JNK和ERK激活以及不同分支的构象要求至关重要 Arrestin介导的信号转导。我们构建了与ASK1、MKK和JNK结合的arrestin-3突变体，但 不促进JNK活性和发现可增强或抑制JNK活性的Arrestin-3短肽 在牢房里。我们将测试信号偏向的拦阻蛋白和arrestin衍生的分子工具的潜力 促进细胞死亡或存活。特异性增加或阻断促凋亡信号的分子工具 在与细胞过度增殖(如癌症)或死亡相关的疾病中具有治疗潜力 (例如，神经退行性疾病)。利用多巴胺D1受体特异性突变体Arrestin-3 提示D_1受体介导的信号转导在L-多巴的行为敏化中起作用 和L多巴诱导的帕金森氏病小鼠模型运动障碍的发展，但其他 GPCRs也助长了这些现象。我们认为，偏向信号的逮捕和 基于阻滞剂的分子工具具有特定的功能，将有助于阐明复杂的 细胞信号和产生新的有效的治疗工具。

PROJECT SUMMARY Arrestins were discovered as negative regulators of G protein-coupled receptor (GPCR) signaling via G proteins. New data show that the free and receptor-bound arrestins initiate signaling through MAP kinases, which regulate cell death, survival, and proliferation. For example, free and receptor-bound arrestin-3 scaffolds ASK1-MKK4/7-JNK1/2/3 cascades, promoting the activation of JNK family kinases. The two non-visual arrestins interact with ~800 different GPCRs in humans. We propose to identify arrestin elements responsible for receptor specificity, and elucidate the structural basis of the assembly of multi-protein signaling complexes (signalosomes) organized by arrestins. Our new crystal structure of arrestin-3 in the presence of an abundant cytoplasmic molecule IP6 revealed receptor-bound-like (active) conformation of arrestin-3. The ability of arrestin-3 to assume this conformation in the absence of GPCRs likely explains receptor-independent scaffolding activity of arrestin-3. We identified arrestin-3 elements critical for JNK and ERK activation, as well as conformational requirements of distinct branches of arrestin-mediated signaling. We constructed arrestin-3 mutants that bind ASK1, MKK and JNK, but do not promote JNK activation and identified short arrestin-3 peptides that enhance or suppress JNK activity in cells. We will test the potential of signaling-biased arrestins and arrestin-derived molecular tools to facilitate cell death or survival. Molecular tools that specifically increase or block pro-apoptotic signaling have therapeutic potential in disorders associated with excessive cell proliferation (e.g., cancer) or death (e.g., neurodegenerative diseases). Using arrestin-3 mutant specific for dopamine D1 receptor we showed that arrestin-mediated signaling from D1 plays a role in the behavioral sensitization to L-DOPA and development of L-DOPA-induced diskinesia in mouse model of Parkinson’s disease, but other GPCRs also contribute to these phenomena. We believe that signaling-biased arrestins and arrestinbased molecular tools with specific functional capability will help elucidate the intricacies of cellular signaling and yield novel potent therapeutic tools.