Protein targeting in vertebrate photoreceptors

脊椎动物光感受器中的蛋白质靶向

• 批准号: 8440782
• 负责人: Sheila A Baker
• 金额: $ 34.43万
• 依托单位: UNIVERSITY OF IOWA
• 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 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8440782
• 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; 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.

描述（由申请人提供）：本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。光感受器的质膜由具有不同蛋白质组成和功能的两个主要隔室组成-外节质膜和内节质膜。这种分离对于光感受器对光产生可靠的电响应是必不可少的。先前的研究表明，某些外节膜蛋白含有特定的靶向信号，以确保它们被正确分选。然而，没有什么是已知的分子和细胞机制负责蛋白质靶向周围的感光细胞的其余部分质膜。在本申请中提出的实验中，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（HCN 1），Na/K-ATP酶和突触素。我们认为HCN 1和Na/K-ATP酶靶向作用的控制机制涉及膜细胞骨架内的一种必需的衔接蛋白-锚蛋白-B。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亚结构域的蛋白质组成。在光感受器中，锚蛋白-B装饰外节外的整个质膜，这使得它成为一个有吸引力的候选者，作为蛋白质靶向进入该域的主要组织者。目的1测试HCN 1与锚蛋白-B结合并且这种相互作用是HCN 1靶向所需的假设。目的2验证了在光感受器中，已知的锚蛋白结合蛋白Na/K-ATP酶与锚蛋白B而不是其他锚蛋白的选择性相互作用是在光感受器中表达的Na/K-ATP酶的特异性α亚基的特征，并且这种选择性相互作用对于Na/K-ATP酶的靶向是必需的。值得注意的是，与锚蛋白-B的相互作用是非常稳定的，而一些蛋白质在它们掺入细胞器如突触囊泡之前瞬时靶向质膜。在目标3中，我们将集中在一个这样的突触囊泡驻留蛋白，突触素。我们已经在突触体蛋白中发现了一种新的靶向序列，并将通过鉴定与该序列结合并调节突触体蛋白靶向的蛋白质来继续这一发现。总之，这些实验将提供两个用于驱动膜蛋白特异性的内段质膜的靶向途径的机制的见解。