Structure-function studies of visual arrestin

描述（由申请人提供）：视觉抑制蛋白是光感受器信号传导的关键参与者，控制信号关闭和光响应恢复的速率。密切相关的非视觉抑制蛋白协调信号和数百种不同的G蛋白偶联受体在几乎每个真核细胞中表达的运输。在这里，我们建议阐明arrestin在光感受器中功能的分子机制，重点是arrestin 1（a.k.a.“视杆”抑制蛋白），其在视杆细胞和视锥细胞中都以高水平表达。使用生物化学和生物物理学的方法相结合，我们建议，以确定视紫红质结合arrestin的构象和形状的arrestin-视紫红质复合物。这将使我们能够理解在与这些“异常”复合物的组成性形成相关的色素性视网膜炎的情况下，抑制蛋白与过度磷酸化视紫红质的复合物如何促成光感受器死亡。我们建议测试是否arrestin优先与单体或二聚体视紫红质相互作用，从而定义的生物相关的arrestin-视紫红质复合物的化学计量。基于我们对arrestin自相关机制的研究，我们建议通过用保留所有其他arrestin功能的自相关受损突变体替换野生型arrestin来阐明这一过程在小鼠感光细胞中的生物学作用。基于我们最初的原理验证实验的成功，我们表明，arrestin突变体具有高亲和力的光激活的非磷酸化视紫红质提高生存和促进光响应恢复在视紫红质磷酸化缺陷的杆，我们建议设计新的“增强”arrestins具有更好的能力，以弥补缺陷的视紫红质磷酸化的先天性视觉障碍的小鼠模型。我们相信，这种“补偿”方法将在所有由功能获得性受体突变体引起的遗传性疾病中具有很高的治疗价值，其中传统的基因替代方法不能“沉默”突变受体的过度信号传导，是无效的。 公共卫生相关性：视觉抑制蛋白是光感受器信号传导的关键参与者，控制信号关闭和光响应恢复的速率。在这里，我们建议阐明arrestin功能的分子机制，重点是arrestin与视紫红质的相互作用，视紫红质结合arrestin的构象和arrestin-rhodopsin复合物的化学计量和形状，以及arrestin在感光细胞中自缔合的机制和生物学作用。我们建议使用这种结构信息来构建“定制设计的”arrestin蛋白，其对光激活的未磷酸化视紫红质具有高亲和力，并测试这些“增强的”突变体补偿先天性视觉障碍小鼠模型中视紫红质磷酸化缺陷的能力。

DESCRIPTION (provided by applicant): Visual arrestins are key players in photoreceptor signaling, governing the rate of signal shutoff and photo response recovery. Closely related non-visual arrestins orchestrate signaling and trafficking of hundreds of different G protein-coupled receptors expressed in virtually every eukaryotic cell. Here we propose to elucidate molecular mechanisms of arrestin function in photoreceptors, focusing on arrestin1 (a.k.a. "rod" arrestin), which is expressed at high level in both rods and cones. Using a combination of biochemical and biophysical methods we propose to determine the conformation of rhodopsin-bound arrestin and the shape of the arrestin-rhodopsin complex. This will allow us to understand how arrestin complexes with hyper- phosphorylated rhodopsin contribute to photoreceptor death in cases of retinitis pigmentosa associated with constitutive formation of these "aberrant" complexes. We propose to test whether arrestin preferentially interacts with monomeric or dimeric rhodopsin, thereby defining the stoichiometry of the biologically relevant arrestin-rhodopsin complex. Based on our studies of the mechanism of arrestin self-association, we propose to elucidate the biological role of this process in photoreceptor cells in mice by replacing wild type arrestin with self-association-impaired mutants that retain all other arrestin functions. Based on the success of our initial proof-of-principle experiments, where we showed that arrestin mutants with high affinity for light-activated unphosphorylated rhodopsin improve the survival and facilitate photo response recovery in rhodopsin phosphorylation-deficient rods, we propose to design new "enhanced" arrestins with better ability to compensate for the defects of rhodopsin phosphorylation in mouse models of congenital visual disorders. We believe that this "compensational" approach will have high therapeutic value in all inherited disorders caused by gain-of-function receptor mutants, where traditional gene replacement approaches, that cannot "silence" excessive signaling by a mutant receptor, are ineffective. PUBLIC HEALTH RELEVANCE: Visual arrestins are key players in photoreceptor signaling, governing the rate of signal shut-off and photo response recovery. Here we propose to elucidate molecular mechanisms of arrestin function, focusing on the arrestin interaction with rhodopsin, the conformation of rhodopsin-bound arrestin and the stoichiometry and shape of the arrestin-rhodopsin complex, as well as on the mechanism and biological role of arrestin self- association in photoreceptor cells. We propose to use this structural information to construct "custom- designed" arrestin proteins with high affinity for light-activated unphosphorylated rhodopsin and test the ability of these "enhanced" mutants to compensate for the defects of rhodopsin phosphorylation in mouse models of congenital visual disorders.