Membrane Targeting by APPL1 and APPL2: Dynamic Scaffolds that Oligomerize and Bind Phosphoinositides

• Published in: Traffic (Copenhagen, Denmark) Vol. 9; no. 2; pp. 215 - 229
• Main Authors: Chial, Heidi J, Wu, Ruping, Ustach, Carolyn V, McPhail, Linda C, Mobley, William C, Chen, Yong Q
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01.02.2008; Blackwell Publishing Ltd
• Subjects: Adaptor Proteins, Signal Transducing; Animals; APPL1; APPL2; BAR domain; Carrier Proteins - genetics; Carrier Proteins - metabolism; Carrier Proteins - physiology; Cell Line, Tumor; Cell Membrane - metabolism; Cytoplasmic Vesicles - metabolism; endocytosis; Golgi Apparatus - metabolism; Humans; Immunoprecipitation; Intracellular Membranes - metabolism; Mice; PH domain; Phosphatidylinositols - metabolism; phosphoinositide binding; Protein Binding; Protein Transport - physiology; PTB domain; RAB5; rab5 GTP-Binding Proteins - genetics; rab5 GTP-Binding Proteins - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; RNA, Small Interfering - genetics; signaling endosome; Transfection; Two-Hybrid System Techniques
• ISSN: 1398-9219; 1600-0854
• DOI: 10.1111/j.1600-0854.2007.00680.x

Human adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1 (APPL1) and adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 2 (APPL2) are homologous effectors of the small guanosine triphosphatase RAB5 that interact with a diverse set of receptors and signaling proteins and are proposed to function in endosome-mediated signaling. Herein, we investigated the membrane-targeting properties of the APPL1 and APPL2 Bin/Amphiphysin/Rvs (BAR), pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains. Coimmunoprecipitation and yeast two-hybrid studies demonstrated that full-length APPL proteins formed homooligomers and heterooligomers and that the APPL minimal BAR domains were necessary and sufficient for mediating APPL-APPL interactions. When fused to a fluorescent protein and overexpressed, all three domains (minimal BAR, PH and PTB) were targeted to cell membranes. Furthermore, full-length APPL proteins bound to phosphoinositides, and the APPL isolated PH or PTB domains were sufficient for in vitro phosphoinositide binding. Live cell imaging showed that full-length APPL-yellow fluorescent protein (YFP) fusion proteins associated with cytosolic membrane structures that underwent movement, fusion and fission events. Overexpression of full-length APPL-YFP fusion proteins was sufficient to recruit endogenous RAB5 to enlarged APPL-associated membrane structures, although APPL1 was not necessary for RAB5 membrane targeting. Taken together, our findings suggest a role for APPL proteins as dynamic scaffolds that modulate RAB5-associated signaling endosomal membranes by their ability to undergo domain-mediated oligomerization, membrane targeting and phosphoinositide binding.