The clathrin adaptor Dab2 recruits EH domain scaffold proteins to regulate integrin ß1 endocytosis.

Endocytic adaptor proteins facilitate cargo recruitment and clathrin-coated pit nucleation. The prototypical clathrin adaptor AP2 mediates cargo recruitment, maturation, and scission of the pit by binding cargo, clathrin, and accessory proteins, including the Eps-homology (EH) domain proteins Eps15 and intersectin. However, clathrin-mediated endocytosis of some cargoes proceeds efficiently in AP2-depleted cells. We found that Dab2, another endocytic adaptor, also binds to Eps15 and intersectin. Depletion of EH domain proteins altered the number and size of clathrin structures and impaired the endocytosis of the Dab2- and AP2-dependent cargoes, integrin ß1 and transferrin receptor, respectively. To test the importance of Dab2 binding to EH domain proteins for endocytosis, we mutated the EH domain-binding sites. This mutant localized to clathrin structures with integrin ß1, AP2, and reduced amounts of Eps15. Of interest, although integrin ß1 endocytosis was impaired, transferrin receptor internalization was unaffected. Surprisingly, whereas clathrin structures contain both Dab2 and AP2, integrin ß1 and transferrin localize in separate pits. These data suggest that Dab2-mediated recruitment of EH domain proteins selectively drives the internalization of the Dab2 cargo, integrin ß1. We propose that adaptors may need to be bound to their cargo to regulate EH domain proteins and internalize efficiently.

FCH domain only-2 organizes clathrin-coated structures and interacts with Disabled-2 for low-density lipoprotein receptor endocytosis.

Clathrin-mediated endocytosis regulates the internalization of many nutrient and signaling receptors. Clathrin and endocytic accessory proteins are recruited to receptors by specific adaptors. The adaptor Disabled-2 (Dab2) recruits its cargoes, including the low-density lipoprotein receptor (LDLR), and mediates endocytosis, even when the major adaptor protein AP2 is depleted. We hypothesized that the accessory proteins normally recruited by AP2 may be recruited by Dab2 if AP2 is absent. We identified one such accessory protein, the F-BAR protein FCH domain only-2 (FCHO2), as a major Dab2-interacting protein. The µ-homology domain (µHD) of FCHO2 binds directly to DPF sequences in Dab2 that also bind AP2. Disrupting the Dab2-FCHO2 interaction inhibited Dab2-mediated LDLR endocytosis in AP2-depleted cells. Depleting FCHO2 reduced the number but increased the size of clathrin structures on the adherent surface of HeLa cells and inhibited LDLR and transferrin receptor clustering. However, LDLR was internalized efficiently by FCHO2-deficient cells when additional time was provided for LDLR to enter the enlarged structures before budding, suggesting that later steps of endocytosis are normal under these conditions. These results indicate FCHO2 regulates the size of clathrin structures, and its interaction with Dab2 is needed for LDLR endocytosis under conditions of low AP2.

S/P and T/P phosphorylation is critical for tau neurotoxicity in Drosophila.

The microtubule-associated protein tau is hyperphosphorylated abnormally in AD and related neurodegenerative disorders. Many phospho epitopes created by proline directed kinases (SP/TP sites) show relative specificity for disease states. To test whether phosphorylation at the disease-associated SP/TP sites affects tau toxicity in vivo, we expressed a form of tau in Drosophila in which all SP/TP sites are mutated to alanine. We find that blocking phosphorylation at SP/TP motifs markedly reduces tau toxicity in vivo. Using phosphorylation-specific antibodies, we identify a positive correlation between increased phosphorylation at disease-associated sites and neurotoxicity. We use the phosphorylation-incompetent version of tau to show that kinase and phosphatase modifiers of tau neurotoxicity, including cdk5/p35, the JNK kinase hemipterous and PP2A act via SP/TP phosphorylation sites. We provide direct evidence in an animal model system to support the role of phosphorylation at SP/TP sites in playing a critical role in tau neurotoxicity.