Cargo sorting during endocytic recycling

内吞再循环对于许多细胞事件至关重要，包括重要营养受体的再循环， 例如转铁蛋白受体和低密度脂蛋白受体，用于配体的循环 摄取，以及整合素从收缩边缘到细胞前缘的重新分布， 介导它们的运动性。尽管有许多记录在案的重要作用，但蛋白质在 内吞再循环，一个关键调节其功能的过程，仍然知之甚少。在 初步研究，我们已经确定ACAP1在货物蛋白的分选中起作用， 在再循环内体的组成性和调节性再循环，其磷酸化提供了一个 解释它如何能够参与这两个过程。此外，我们还发现， ACAP1的表达诱导了内体的包被。因此，在目标1中，我们将提供更明确的测试， 使用产生转运载体的重构系统，ACAP1是否作为外壳蛋白发挥功能 从回收内体，其次是方法，这将证明，要么单独的ACAP1的功能， 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， 因为调节运输中的关键环节涉及与运输接口的信号过程 在这个过程中，我们将鉴定预测直接磷酸化ACAP1的激酶。在目标3中，我们 鉴定由ACAP1识别的用于组成性再循环的分选信号，我们还将鉴定分选信号 被ACAP1认可用于监管回收，使用整合素β 1作为模型系统。这一发现将 然后使我们能够进一步阐明ACAP1是如何能够参与组成性和调节性的， 回收利用。此外，整合素β 1中的再循环分选信号的鉴定将允许我们测试更多 明确整合素再循环是否在细胞迁移中起关键作用，从而证明， 更好地理解内吞再循环对理解其他细胞事件具有重要意义。

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 thatoverexpression 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 betal 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 betal 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.