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Membrane Targeting by APPL1 and APPL2: Dynamic Scaffolds that Oligomerize and Bind Phosphoinositides
Author(s) -
Chial Heidi J.,
Wu Ruping,
Ustach Carolyn V.,
McPhail Linda C.,
Mobley William C.,
Chen Yong Q.
Publication year - 2008
Publication title -
traffic
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.677
H-Index - 130
eISSN - 1600-0854
pISSN - 1398-9219
DOI - 10.1111/j.1600-0854.2007.00680.x
Subject(s) - pleckstrin homology domain , signal transducing adaptor protein , microbiology and biotechnology , biology , fusion protein , leucine zipper , endosome , phosphotyrosine binding domain , amphiphysin , membrane protein , biochemistry , membrane , signal transduction , endocytosis , sh2 domain , receptor , peptide sequence , proto oncogene tyrosine protein kinase src , dynamin , intracellular , gene , recombinant dna
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.