Mechanisms of Endocytic Recycling

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 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.