Cargo sorting during endocytic recycling

描述(申请人提供)：内吞循环对许多细胞事件至关重要，包括重要的营养受体的循环，如转铁蛋白受体和低密度脂蛋白受体，用于反复的配体摄取，以及整合素从细胞的收缩边缘到前沿的重新分配，以调节其运动。尽管有许多重要的作用被证明，但蛋白质在内吞循环过程中是如何分类的，一个关键地调节其功能的过程仍然知之甚少。在初步研究中，我们已经确定ACAP1在货物蛋白的分类中发挥作用，这是循环内体结构性和受控循环的例证，其磷酸化为它如何能够参与这两个过程提供了解释。此外，我们还发现ACAP1的过表达诱导了内体的包被。因此，在目标1中，我们将使用重建系统提供更明确的测试，以确定ACAP1是否作为外壳蛋白发挥功能，该重建系统从循环内涵体产生运输载体，随后将证明ACAP1单独作为 一种外壳蛋白，或它与其他蛋白质一起作为外壳复合体的一部分发挥作用。在目标2中，由于调控转运中的关键联系涉及信号转导过程与转运过程的接口，我们将确定被预测为直接磷酸化ACAP1K(S)。在目标3中，由于我们已经识别了ACAP1识别的用于结构性循环的分选信号，我们还将以整合素Beta1为模型系统，识别ACAP1识别的用于受调控循环的分选信号(S)。这一发现将使我们能够进一步阐明ACAP1如何能够参与结构性和受监管的回收。此外，整合素Beta1中回收分选信号(S)的识别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发挥关键作用，从而表明更好地理解细胞内循环对理解其他细胞事件具有重要意义。

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.