Cargo sorting during endocytic recycling

内吞回收过程中的货物分类

• 批准号: 6968420
• 负责人: VICTOR W HSU
• 金额: $ 31.49万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6968420
• 关键词: biological signal transduction; cell migration; cell motility; electron microscopy; endocytosis; enzyme activity; integrins; phosphorylation; protein binding; protein kinase; protein reconstitution; protein structure function; protein transport; site directed mutagenesis; transfection; vesicle /vacuole

DESCRIPTION (provided by applicant): Endocytic recycling is critical for many cellular events, including the recycling of important nutrient receptors, such as the transferrin receptor and the low-density lipoprotein receptor, for reiterative rounds of ligand uptake, and also the redistribution of integrins from the retracting edges to the leading edge of cells to mediate their motility. Despite these many documented important roles, how proteins are sorted during endocytic recycling, a process that critically regulates their function remains poorly understood. In preliminary studies, we have identified ACAP1 to function in the sorting of cargo proteins that exemplify constitutive and regulated recycling at the recycling endosome, with its phosphorylation providing an explanation for how it is able to participate in both processes. Moreover, we have found that overexpression of ACAP1 induces the coating of endosomes. Thus, in Aim 1, we will provide a more definitive test for whether ACAP1 functions as a coat protein using a reconstitution system that generates transport carriers from the recycling endosome, followed by approaches which will either prove that ACAP1 alone functions as a coat protein or that it functions in conjunction with other proteins as part of coat complex. In Aim 2, because the critical nexus in regulated transport involves the signaling process interfacing with the transport process, we will identify kinase(s) predicted to directly phosphorylate ACAP1. In Aim 3, as we have identified sorting signals recognized by ACAP1 for constitutive recycling, we will also identify sorting signal(s) recognized by ACAP1 for regulated recycling, using integrin beta1 as the model system. This finding will then enable us to further elucidate how ACAP1 is able to participate in both constitutive and regulated recycling. Moreover, the identification of recycling sorting signal(s) in integrin beta1 will allow us to test more definitively whether integrin recycling plays a critical role in cell migration, and thereby demonstrating that a better understanding of endocytic recycling has important implications to understanding other cellular events.

描述（由申请人提供）：内吞再循环对于许多细胞事件至关重要，包括重要营养受体（如转铁蛋白受体和低密度脂蛋白受体）的再循环，用于配体摄取的循环，以及整合素从细胞收缩边缘到前缘的再分布，以介导其运动性。尽管有这些许多记录的重要作用，蛋白质如何在内吞再循环过程中进行分类，这是一个关键调节其功能的过程，但仍然知之甚少。在初步研究中，我们已经确定了ACAP1在货物蛋白的分选中起作用，这些货物蛋白在回收内体中具有组成性和调节回收的功能，其磷酸化为它如何能够参与这两个过程提供了解释。此外，我们发现过表达ACAP1诱导内体的包被。因此，在目标1中，我们将提供一种更确定的测试，以确定ACAP1是否作为外壳蛋白发挥作用，使用从再循环内体产生转运载体的重建系统，然后是证明ACAP1单独作为外壳蛋白发挥作用的方法。 外壳蛋白或它与其他蛋白质一起作为外壳复合物的一部分起作用。在目标2中，由于调节转运中的关键关系涉及与转运过程相互作用的信号传导过程，因此我们将鉴定预测直接磷酸化ACAP1的激酶。在目标3中，由于我们已经鉴定了由ACAP1识别的用于组成性再循环的分选信号，我们还将鉴定由ACAP1识别的用于调节性再循环的分选信号，使用整联蛋白β 1作为模型系统。这一发现将使我们能够进一步阐明ACAP1如何能够参与组成性和调节性再循环。此外，整合素β 1中回收分选信号的鉴定将使我们能够更明确地测试整合素回收是否在细胞迁移中起关键作用，从而证明更好地理解内吞回收对理解其他细胞事件具有重要意义。