Protein targeting in vertebrate photoreceptors

脊椎动物光感受器中的蛋白质靶向

• 批准号: 7860738
• 负责人: Sheila A Baker
• 金额: $ 1.96万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7860738
• 关键词: Adaptor Signaling Protein; Ankyrins; Binding; Binding Proteins; Biological Assay; Cell membrane; Cells; Co-Immunoprecipitations; Cytoskeleton; Degenerative Disorder; Destinations; Disease; Elements; Ensure; Goals; Light; Maintenance; Mechanics; Mediating; Membrane; Membrane Proteins; Molecular; Mus; Na(+)-K(+)-Exchanging ATPase; Organelles; Output; Pathway interactions; Photoreceptors; Presynaptic Terminals; Process; Protein Binding; Proteins; Rana; Reading; Recombinant Proteins; Rest; Retina; Retinal Degeneration; Series; Signal Transduction; Sorting - Cell Movement; Synapses; Synaptic Vesicles; Synaptophysin; Testing; Therapeutic Intervention; Transgenic Organisms; Vertebrate Photoreceptors; Work; base; cyclic-nucleotide gated ion channels; insight; knowledge base; mutant; novel; polarized cell; protein transport; public health relevance; research study; response

DESCRIPTION (provided by applicant): The overall goal of this proposal is to uncover the mechanisms of protein trafficking utilized by the vertebrate photoreceptor to establish and maintain its unique subcellular compartmentalization. The plasma membrane of the photoreceptor consists of two major compartments with distinct protein composition and function - the outer segment plasma membrane and the inner segment plasma membrane. This separation is essential for photoreceptors to produce a reliable electrical response to light. Previous studies revealed that certain outer segment membrane proteins contain specific targeting signals to ensure they are properly sorted. However, nothing is known about the molecular and cellular mechanisms responsible for protein targeting to the plasma membrane surrounding the rest of the photoreceptor cell. In the experiments proposed in this application we will investigate the mechanisms controlling the targeting of three different proteins to the inner segment plasma membrane: the hyperpolarization-activated cyclic nucleotide-gated channel (HCN1), Na/K-ATPase and synaptophysin. We propose that the mechanism controlling the targeting of both HCN1 and Na/K-ATPase involves ankyrin-B, an essential adaptor protein within the membrane cytoskeleton. The membrane cytoskeleton lines the plasma membrane of all cells; it provides mechanical stability and regulates the protein composition of membrane subdomains. In photoreceptors, ankyrin-B decorates the entire plasma membrane outside the outer segment, which makes it an attractive candidate for serving as a master organizer of protein targeting into this domain. Aim 1 tests the hypothesis that HCN1 binds to ankyrin-B and that this interaction is required for the targeting of HCN1. Aim 2 tests the hypothesis that in photoreceptors the selective interaction of Na/K-ATPase, a known ankyrin binding protein, with ankyrin-B but not other ankyrins is a feature of the specific alpha subunit of Na/K-ATPase expressed in photoreceptors and that this selective interaction is necessary for the targeting of Na/K-ATPase. Notably, interactions with ankyrin-B are very stable, whereas some proteins are targeted to the plasma membrane transiently prior to their incorporation into organelles such as synaptic vesicles. In Aim 3 we will focus on one such synaptic vesicle resident protein, synaptophysin. We have found a novel targeting sequence within synaptophysin and will pursue this finding by identifying the protein(s) that bind to this sequence and regulate the targeting of synaptophysin. Together, these experiments will provide mechanistic insight into two targeting pathways used to drive membrane proteins specifically to the inner segment plasma membrane. PUBLIC HEALTH RELEVANCE: This work is relevant to understanding the molecular bases of blinding degenerative diseases of the retina, often characterized by disorders in photoreceptor protein trafficking or the maintenance of cellular compartmentalization. Elucidating the mechanisms controlling these processes is essential for developing therapeutic interventions.

描述（由申请人提供）：该提案的总体目标是揭示脊椎动物光感受器用于建立和维持其独特的亚细胞区室化的蛋白质运输机制。光感受器的质膜由两个具有不同蛋白质组成和功能的主要区室组成——外段质膜和内段质膜。这种分离对于感光器产生可靠的光电响应至关重要。先前的研究表明，某些外段膜蛋白含有特定的靶向信号，以确保它们被正确分类。然而，对于蛋白质靶向感光细胞其余部分周围质膜的分子和细胞机制一无所知。在本申请提出的实验中，我们将研究控制三种不同蛋白质靶向内段质膜的机制：超极化激活的环核苷酸门控通道（HCN1）、Na/K-ATP酶和突触素。我们提出控制 HCN1 和 Na/K-ATPase 靶向的机制涉及锚蛋白-B，它是膜细胞骨架内的一种重要接头蛋白。膜细胞骨架排列在所有细胞的质膜上；它提供机械稳定性并调节膜子域的蛋白质组成。在光感受器中，锚蛋白-B 装饰外节之外的整个质膜，这使其成为靶向该结构域的蛋白质主要组织者的有吸引力的候选者。目标 1 测试 HCN1 与锚蛋白-B 结合的假设，并且这种相互作用是 HCN1 靶向所必需的。目标 2 检验以下假设：在光感受器中，Na/K-ATP 酶（一种已知的锚蛋白结合蛋白）与锚蛋白-B（而非其他锚蛋白）的选择性相互作用是光感受器中表达的 Na/K-ATP 酶的特定 α 亚基的一个特征，并且这种选择性相互作用对于 Na/K-ATP 酶的靶向是必要的。值得注意的是，与锚蛋白-B 的相互作用非常稳定，而一些蛋白质在掺入突触小泡等细胞器之前会短暂地靶向质膜。在目标 3 中，我们将重点关注一种这样的突触小泡驻留蛋白：突触素。我们在突触素内发现了一个新的靶向序列，并将通过鉴定与该序列结合并调节突触素靶向的蛋白质来追求这一发现。总之，这些实验将提供对用于将膜蛋白特异性驱动至内段质膜的两种靶向途径的机制见解。 公共健康相关性：这项工作与了解致盲性视网膜退行性疾病的分子基础相关，这些退行性疾病通常以光感受器蛋白运输障碍或细胞区室化维持为特征。阐明控制这些过程的机制对于制定治疗干预措施至关重要。