Cargo sorting during endocytic recycling

描述（由申请人提供）：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缘的再分配，以调节其运动性。尽管有许多文献记载的重要作用，但在内吞循环过程中蛋白质是如何分类的，这是一个关键的调节其功能的过程，人们对蛋白质的分类仍然知之甚少。在初步研究中，我们已经确定ACAP1在货物蛋白的分选中发挥作用，这些蛋白质在回收内体中体现了构成性和规范性的回收，其磷酸化解释了它如何能够参与这两个过程。此外，我们还发现ACAP1的过表达诱导了核内体的包被。因此，在Aim 1中，我们将使用从回收内体产生运输载体的重构系统对ACAP1是否作为外壳蛋白进行更明确的测试，然后采用方法证明ACAP1单独作为外壳蛋白

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.