Mechanisms of Endocytic Recycling

 描述(由申请人提供)：内吞循环对许多生理事件至关重要，包括营养吸收、细胞信号、极性以及细胞黏附和迁移。近年来，我们通过确定在这一过程中起作用的第一个涂层复合体，促进了对细胞内循环的基本理解。该复合体由连接到ACAP1(ArfGap with Coil-Coil，Ankyrin Repeat and PH域1)的新型适配器上的笼蛋白组成。我们还鉴定了ARF6(ADP-核糖化因子6)来调节这个外壳复合体，而Grp1(3-磷酸肌醇1的通用受体)则作为鸟嘌呤核苷酸交换因子(Gef)。最近，我们对这些因素的研究发现了重要的新见解，我们建议进一步深入研究。首先，我们最近寻求了一种将单颗粒冷冻电子显微镜与蛋白质结晶学相结合的尖端方法。这种方法为ACAP1如何弯曲细胞膜提供了意想不到的新见解。因此，我们建议应用这种结合的方法来研究ARF6如何弯曲膜，并研究ARF6如何与ACAP1合作来传递膜曲率。其次，我们还发现Akt通过一种非激酶功能促进内吞循环。因此，我们将探讨这一新角色的三种可能性：i)作为货物适配器促进ACAP1与货物的结合，ii)调节ACAP1的GTP酶激活蛋白(GAP)活性，或iii)调节ACAP1弯曲膜的能力。第三，我们还发现了对Grp1调控的新见解，这涉及到其PH(Pleckstrin Homology)结构域的关键残基的磷酸化，导致Grp1靶向循环内体而不是质膜。我们还确定了促进这一转变的其他因素。因此，我们将阐明这些不同之处 可以协调目标机制，从而有助于更好地理解如何以复杂的方式管理区域合作伙伴关系全球环境基金，以确保适当地启动运输途径。由于内吞循环是许多关键生理事件的基础，我们预计我们的研究不仅将促进对运输机制的基本理解，还将深入了解内吞循环如何对健康和疾病状态都至关重要。

 DESCRIPTION (provided by applicant): Endocytic recycling is critical for many physiologic events, including nutrient uptake, cell signaling, polarity, and cell adhesion and migration. In recent years, we have advanced a fundamental understanding of endocytic recycling by identifying the first coat complex to act in this process. This complex is composed of clathrin coupled to a novel adaptor, known as ACAP1 (Arfgap with Coil-coil, Ankyrin repeat and PH domain 1). We have also identified ARF6 (ADP-Ribosylation Factor 6) to regulate this coat complex, with Grp1 (General receptor for 3-phosphoinositides 1) acting as the guanine nucleotide exchange factor (GEF). More recently, our studies on these factors have uncovered significant new insights, which we propose to pursue in further detail. First, we have recently pursued a cutting-edge approach that combines single-particle cryo-electron microscopy with protein crystallography. This approach has shed unexpected new insight into how ACAP1 bends membrane. Thus, we propose to apply this combined approach to study how ARF6 bends membrane, and also examine how ARF6 cooperates with ACAP1 to impart membrane curvature. Second, we have also uncovered that Akt promotes endocytic recycling through a non-kinase function. Thus, we will examine three possibilities for this novel role: i) acting as a cargo adaptor to promote cargo binding by ACAP1, ii) regulating the GTPase activating protein (GAP) activity of ACAP1, or iii) modulating the ability of ACAP1 to bend membrane. Third, we have also uncovered new insights into the regulation of Grp1, which involves the phosphorylation of a key residue in its PH (Pleckstrin Homology) domain, resulting in Grp1 being targeted to the recycling endosome rather than the plasma membrane. We have also identified additional factors that promote this switch. Thus, we will elucidate how these different targeting mechanisms may be coordinated, and thereby contributing to a better understanding of how ARF GEFs are regulated in complex ways to ensure that a transport pathway is properly initiated. As endocytic recycling underlies many key physiologic events, we anticipate that our studies will not only advance a basic understanding of transport mechanisms, but also shed insights into how endocytic recycling is critical for both health and disease states.