EFA6A, a guanine nucleotide exchange factor for Arf6, interacts with sorting nexin‐1 and regulates neurite outgrowth

Abstract

The membrane trafficking and actin cytoskeleton remodeling mediated by ADP ribosylation factor 6 (Arf6) are functionally linked to various neuronal processes including neurite formation and maintenance, neurotransmitter release, and receptor internalization. EFA6A is an Arf6-specific guanine nucleotide exchange factor that is abundantly expressed in the brain. In this study, we identified sorting nexin-1 (SNX1), a retromer component that is implicated in endosomal sorting and trafficking, as a novel interacting partner for EFA6A by yeast two-hybrid screening. The interaction was mediated by the Cterminal region of EFA6A and a BAR domain of SNX1, and further confirmed by pull-down assay and immunoprecipitation from mouse brain lysates. In situ hybridization analysis demonstrated the widespread expression of SNX1 in the mouse brain, which overlapped with the expression of EFA6A in the forebrain. Immunofluorescent analysis revealed the partial colocalization of EFA6A and SNX1 in the dendritic fields of the hippocampus. Immunoelectron microscopic analysis revealed the overlapping subcellular localization of EFA6A and SNX1 at the post-synaptic density and endosomes in dendritic spines. In Neuro-2a neuroblastoma cells, expression of either EFA6A or SNX1 induced neurite outgrowth, which was further enhanced by co-expression of EFA6A and SNX1. The present findings suggest a novel mechanism by which EFA6A regulates Arf6-mediated neurite formation through the interaction with SNX1. Keywords: ADP ribosylation factor, dendritic spine, guanine nucleotide exchange factor, membrane traffic, post-synaptic density. The endocytic system comprises various membrane compartments including early endosomes, recycling endosomes, late endosomes, lysosomes, and the trans-Golgi network (Maxfield and McGraw 2004). The endosomal trafficking of membrane components at appropriate places and timing is intimately linked to a variety of neuronal functions including neuronal polarity, migration, neurite formation, synaptic connection, neurotransmitter release, and receptor surface expression (Kennedy and Ehlers 2006;Schmidt and Haucke 2007;Kawauchi 2012;Yap and Winckler 2012). During neurite formation, the polarized delivery of membrane components to growing neurite tips through the endocytic system is required for vectorial neurite outgrowth and extension (Tang 2008;Sann et al. 2009). Once synaptic connections are established, activity-dependent changes in synaptic efficiency and structures are regulated by endosomal trafficking of membrane components such as a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-type glutamate receptors (AMPARs) from dendritic recycling endosomes to the post-synaptic spine membrane, which underlies a fundamental mechanism for synaptic plasticity (Hanley 2010; Anggono and Huganir 2012). Various small GTPases, particularly the ADP ribosylation factor (Arf) and Rab families, have been recognized as critical regulators of Received September 5, 2013; revised manuscript received October 9, 2013; accepted November 4...

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