ARF regulators in endocytic transport

描述（由申请人提供）：adp -核糖基化因子（ARFs）调节外壳蛋白，作为耦合囊泡形成和囊泡运输货物分选的核心细胞机制。反过来，ARF小gtpase由催化ARF活化的鸟嘌呤核苷酸交换因子（gef）和催化ARF失活的gtpase活化蛋白（gap）调节。在上一个资助期内，我们一直在研究三种ARF调节因子在内吞运输中的作用。首先，我们确定了一种称为ACAP1的ARF GAP，它作为一种新型网格蛋白外壳复合体的核心适配器，用于内吞循环。我们还发现，ACAP1参与了本构回收和管制回收，其货物绑定解释了如何完成这两种类型的运输。因此，我们建议采用生物化学和结构相结合的方法进一步阐明ACAP1在货物分拣中的作用。其次，我们已经确定Grp1是作用于ARF6的GEF，用于葡萄糖转运体4型（glut4）的内吞循环，这一过程受胰岛素调节。因此，我们建议采用形态学和生化相结合的方法来阐明胰岛素诱导的调节glut4循环的信号传导如何靶向这种GEF。第三，我们发现ARFGAP1是由转铁蛋白受体内吞作用定义的网格蛋白ap2依赖性转运的一个子集所必需的。因此，我们将进一步阐明这一作用，研究ARFGAP1如何参与货物分选和囊泡形成，并质疑其GAP活性的作用。我们预计这些结果将为内吞作用和循环机制提供关键见解。此外，由于控制这些转运途径的机制缺陷可能导致和/或促成心血管、免疫、神经和致癌疾病，我们进一步预计，我们的结果将对理解和治疗人类疾病产生广泛的影响。

DESCRIPTION (provided by applicant): ADP-Ribosylation Factors (ARFs) regulate coat proteins, which act as the core cellular machinery in coupling vesicle formation and cargo sorting for vesicular transport. The ARF small GTPases are, in turn, regulated by guanine nucleotide exchange factors (GEFs) that catalyze ARF activation and GTPase-activating proteins (GAPs) that catalyze ARF deactivation. In the last funding period, we have been studying how three ARF regulators act in endocytic transport. First, we have identified an ARF GAP, known as ACAP1, to act as the core adaptor in a novel clathrin coat complex for endocytic recycling. We have also found that ACAP1 participates in both constitutive and regulated recycling, with its cargo binding explaining how both types of transport can be accomplished. Thus, we propose to further elucidate how ACAP1 acts in cargo sorting by taking combined biochemical and structural approaches. Second, we have identified Grp1 as the GEF that acts on ARF6 for the endocytic recycling of the glucose transporter type 4 (glut4), a process that is regulated by insulin. Thus, we propose to take combined morphologic and biochemical approaches to elucidate how this GEF may be targeted by insulin-induced signaling that regulates glut4 recycling. Third, we have found that ARFGAP1 is required for a subset of clathrin AP2-dependent transport, as defined by the endocytosis of transferrin receptor. Thus, we will further elucidate this role, examining how ARFGAP1 participates in cargo sorting and vesicle formation and also interrogating the role of its GAP activity. We anticipate that these results will shed key insights into mechanisms of endocytosis and recycling. Moreover, because defects in mechanisms that govern these transport pathways can cause and/or contribute to cardiovascular, immunologic, neurologic and oncogenic diseases, we further anticipate that our results will have broad ramifications to understanding and treating human diseases. PUBLIC HEALTH RELEVANCE: The function of proteins is critically regulated by their localization. This localization is achieved in part by transport pathways that act as highways within the cell. We propose to understand how key components of these pathways function in regulating the distribution of proteins on the cell surface. This understanding will be broadly applicable to understanding human diseases, as many are now appreciated to arise because of defects in protein transport within the cell.