Targeted Engineering of Designer Arrestins to Regulate Cell Signaling

项目摘要 抑制蛋白是G蛋白偶联受体（GPCR）信号的负调节因子，通过G proteins.新的数据表明，游离和受体结合的抑制蛋白通过MAP启动信号传导， 激酶，其调节细胞死亡、存活和增殖。例如，自由和受体结合 抑制蛋白-3支架ASK 1-MKK 4/7-JNK 1/2/3级联，促进JNK家族激酶的活化。 这两种非视觉抑制蛋白与人类约800种不同的GPCR相互作用。我们建议确定 负责受体特异性的arrestin元件，并阐明组装的结构基础 由抑制蛋白组织的多蛋白信号复合物（信号体）。我们的新晶体结构 抑制蛋白-3在丰富的细胞质分子IP 6的存在下显示受体结合样（活性） arrestin-3的构象。抑制蛋白-3在GPCR不存在下呈现这种构象的能力 可能解释了抑制蛋白-3的受体非依赖性支架活性。我们鉴定了arrestin-3元件 对于JNK和ERK激活以及不同分支的构象要求至关重要。 抑制蛋白介导的信号传导。我们构建了结合ASK 1、MKK和JNK的arrestin-3突变体，但它们不能结合。 不促进JNK活化，并且鉴定了增强或抑制JNK活性的短抑制蛋白-3肽 在细胞中。我们将测试信号偏置抑制蛋白和抑制蛋白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传导的分子工具 在与过度细胞增殖相关的疾病中具有治疗潜力（例如，癌症）或死亡 (e.g.,神经变性疾病）。使用多巴胺D1受体特异性抑制蛋白-3突变体， 显示来自D1的抑制蛋白介导的信号传导在对L-DOPA的行为敏化中起作用 和帕金森病小鼠模型中L-DOPA诱导的运动障碍的发展，但其他 GPCR也促成了这些现象。我们相信信号偏差逮捕和 具有特定功能的基于抑制蛋白的分子工具将有助于阐明 细胞信号传导并产生新的有效治疗工具。

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.