Membrane targeting by APPL1 and APPL2: dynamic scaffolds that oligomerize and bind phosphoinositides.
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.
Department of Cancer Biology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157, USA. hchial@stanford.edu
Heidi J
HJ
Chial
Ruping
R
Wu
Carolyn V
CV
Ustach
Linda C
LC
McPhail
William C
WC
Mobley
Yong Q
YQ
Chen
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D007425:Intracellular Membranes / Q000378:metabolism; D051379:Mice; D010716:Phosphatidylinositols / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D020798:Two-Hybrid System Techniques; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism
C464801:APPL1 protein, human; C528555:APPL2 protein, human; D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D010716:Phosphatidylinositols; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D020696:rab5 GTP-Binding Proteins