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使用的蛋白质运输机制，以建立和维护其独特的亚细胞隔室化。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隔室组成 - 外部段质膜和内部段质膜。这种分离对于光感受器产生对光的可靠电响应至关重要。先前的研究表明，某些外部段膜蛋白包含特定的靶向信号，以确保正确分类它们。然而，关于负责靶向围绕光感受器细胞的质膜的蛋白质的分子和细胞机制一无所知。在本应用程序中提出的实验中，我们将研究控制三种不同蛋白质靶向内部段质膜的机制：高极化的环状核苷酸门控通道（HCN1），Na/k-ATPase和Synaptophophin。我们提出，控制HCN1和Na/K-ATPase靶向的机制涉及Ankyrin-B，Ankyrin-B是膜细胞骨架中必不可少的衔接蛋白。膜细胞骨架在所有细胞的质膜上排成一条；它提供机械稳定性并调节膜亚域的蛋白质组成。在光感受器中，Ankyrin-B在外部段外装饰了整个质膜，这使其成为成为蛋白质靶向该域的主要组织者的有吸引力的候选者。 AIM 1检验了HCN1与Ankyrin-B结合的假设，并且这种相互作用是HCN1靶向所必需的。 AIM 2检验了以下假设：在光感受器中，Na/k-ATPase（一种已知的Ankyrin结合蛋白）与Ankyrin-b（而非其他Ankyrins）的选择性相互作用是在光感受器中表达的Na/k-atpase的特定α亚基的特征，并且是在光感受器中表达的特定特征。值得注意的是，与Ankyrin-B的相互作用非常稳定，而某些蛋白质在将其掺入细胞器（例如突触囊泡）之前，将其靶向质膜。在AIM 3中，我们将重点放在一种此类突触囊泡蛋白突触蛋白上。我们在突触素中发现了一个新颖的靶向序列，并通过识别与该序列结合并调节突触素蛋白的靶向的蛋白质来追求这一发现。这些实验将共同提供针对两种用于驱动膜蛋白的靶向途径的机械洞察力。 公共卫生相关性：这项工作与理解视网膜盲型退行性疾病的分子碱基有关，通常以光感受器蛋白运输或维持细胞分室化的疾病为特征。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。