Arrestin interactions with non-receptor binding partners

描述(申请人提供)：阻滞素最初被描述为通过G蛋白对G蛋白偶联受体(GPCR)信号的负面调节。新的数据显示，自由和受体结合的拦阻蛋白通过MAP激酶启动信号传递，MAP激酶调节细胞的死亡、存活和增殖。特别是，游离和受体相关的arrestin-3支架 ASK1-MKK-JNK级联反应，促进JNK活化。在这里，我们建议用生化和生物物理的方法来阐明依赖arrestin激活促凋亡的JNK家族激酶及其激活剂MKK4/7的结构基础。Arrestin-3组成的多蛋白质信号复合体(信号体)组装的分子机制将被建立，并将识别对JNK激活至关重要的arrestin-3残基。基于这一信息，将构建与ASK1、MKK和JNK结合但不促进JNK激活的arrestin-3突变体。Arrestin突变体激活JNK的能力急剧下降，我们已经有几个JNK，以保护细胞免受侮辱并延长它们的生存时间，这一潜力将得到测试。我们还构建了arrestin-3突变体，它比亲本野生型arrestin-3更有效地激活JNKs。我们建立了依赖arrestin-3的JNK激活在细胞存活中起关键作用的范例。我们将测试过度活跃的arrestin-3突变体促进细胞死亡的能力。我们发现，非活性突变体将JNKs和上游激酶结合在非生产性复合体中，从而以显性-负向的方式发挥作用。我们将测试这些突变体保护细胞并延长其存活时间的潜力。特异性增加或阻断促凋亡信号的分子TOOS在疾病中具有治疗潜力 与细胞过度增殖(如癌症)或死亡(如神经退行性疾病)有关。

DESCRIPTION (provided by applicant): Arrestins were first described 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. In particular, both free and receptor- associated arrestin-3 scaffolds ASK1-MKK-JNK cascade, promoting JNK activation. Here we propose to elucidate the structural basis of arrestin-dependent activation of pro-apoptotic JNK family kinases and their activators MKK4/7 using biochemical and biophysical methods. The molecular mechanisms of the assembly of multi-protein signaling complexes (signalosomes) organized by arrestin-3 will be established, and arrestin-3 residues critical for JNK activation will be identified. Based on ths info, arrestin-3 mutants that bind ASK1, MKK and JNK, but do not promote JNK activation will be constructed. The potential of arrestin mutants with dramatically reduced ability to activate JNKs, several of which we already have, to protect cells against insults and prolong their survival will be tested. We have also constructed arrestin-3 mutants that activate JNKs more efficiently than parental wild type arrestin-3. We established the paradigm where arrestin-3-dependent JNK activation plays key role in cell survival. We will test the ability of hyperactive arrestin-3 mutants to facilitate cell death. We showed that inactive mutants tie up JNKs and upstream kinases in unproductive complexes, thereby acting in dominant- negative manner. We will test the potential of these mutants to protect cells and prolong their survival. Molecular toos 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).