Our results are summarized in and suggest the following model: EH domain proteins regulate the number and size of CCSs and the internalization, but not recruitment, of Dab2-dependent and Dab2-independent cargoes, integrin β1 and TfnR. EH domain proteins are recruited to CCSs by AP2, the NPF sequences in Dab2, and other accessory proteins. If Dab2 is absent, more AP2 enters CCSs, and the content of EH domain proteins is unaffected. However, cargo internalization may depend on specific adaptor–EH domain protein interactions. A Dab2 mutant that cannot bind EH domain proteins (p96 NPF1-5*) acts as a dominant negative, slightly reducing the recruitment of EH domain proteins to CCSs and inhibiting integrin β1 internalization. Internalization of TfnR from a different set of CCSs was unaffected. We propose that specific receptor–adaptor–EH domain protein complexes are required for efficient endocytosis. In addition, the number of clathrin structures with short half-life is reduced in Dab2-deficient cells or cells reconstituted with the p96 NPF1-5* mutant. We speculate that the Dab2–EH domain protein complex may also have a role in determining whether short-lived clathrin structures grow or abort.
FIGURE 6: Summary of results and proposed model. Top, summary of results taken from , Supplemental Figure S2, and , , and =, same as in control; upward arrow, increased; downward arrow, decreased. Bottom, model. Dab2 and AP2 are (more ...)
We found that Eps15 and Itsn bind to NPF sequences in Dab2 (). Whereas NPF is the core of most EH-binding peptides, individual EH domains prefer specific surrounding sequences. For example, the second EH domain of Eps15 shows a slight preference for alanine at +1 (NPFA), and the first EH domain of Itsn is specific for NPFXCOOH
at the C-terminus of a protein (Paoluzi et al., 1998
; Yamabhai et al., 1998
). The fifth NPF motif of Dab2 is the most highly conserved across evolution and fits both these requirements; it lies in the sequence NPFACOOH
. This may explain why the C-terminal region of Dab2 binds Eps15 and Itsn more strongly than the central region (). In addition, Eps15 and Itsn form heterodimers, so they may bind cooperatively or competitively (Sengar et al., 1999
EH domain proteins in CCSs may act as scaffolds to bring in additional endocytic accessory proteins, including dynamin, synaptojanin, stonin, synaptotagmin, SHIP2, FCHO2, and epsin (Miliaras and Wendland, 2004
). We found that depleting EH domain proteins from HeLa cells had no effect on CCPs on the dorsal surface but significantly decreased the number of pits and plaques on the ventral surface. The median size of plaques, but not pits, increased significantly, suggesting a role in CCS nucleation and growth (). It is possible that EH domain proteins help recruit membrane curvature–sensing proteins, like FCHO2, epsin, and endophilin, to form CCPs. In the absence of EH domain proteins, clathrin structures still form, but they tend to be flat plaques rather than curved CCPs.
Our results differ from a report that depletion of EH domain proteins inhibited all clathrin-coated structures (Henne et al., 2010
). However, these authors used BSC1 cells, and the functions of EH domain proteins may depend on the cell type. Unlike HeLa cells, BSC1 cells have neither Dab2 nor plaques, so the presence of Dab2 in HeLa cells may permit CCS nucleation in the absence of EH domain proteins (Ehrlich et al., 2004
; Saffarian et al., 2009
; Mettlen et al., 2010
). Alternatively, HeLa cells may express additional EH domain proteins besides Eps15, Eps15R, Itsn1, and Itsn2 that can also nucleate CCSs. It is possible that complete removal of the entire suite of endocytic EH domain proteins from HeLa cells would also abolish all clathrin-containing structures, as reported in BSC1 cells.
Internalization of Dab2-dependent and Dab2-independent cargoes (integrin β1 and TfnR, respectively) was inhibited in EH-depleted HeLa cells (). Decreased internalization of TfnR may be an indirect consequence of the decreased numbers of pits and plaques and the increased plaque size on the ventral surface of the cell ( and ). Plaques have longer lifetimes (2–16 min) than CCPs (~60 s) (Saffarian et al., 2009
), so internalization from the larger structures may be inhibited. However, this explanation is unlikely to account for the decreased internalization of integrin β1, since integrin β1 is internalized from the dorsal surface, where the number and size of CCPs was unaltered (; Teckchandani et al., 2009
). Therefore it is possible that EH domain proteins may directly regulate internalization of integrin β1. This conclusion is supported by use of a Dab2 mutant that does not bind EH proteins. This mutant fails to support integrin β1 internalization, but TfnR internalization is normal ( and ). Separate regulation may be possible because integrin β1 and TfnR do not significantly colocalize, suggesting that they internalize from different CCSs (). This separation may have arisen because integrin β1 and TfnR follow different endocytic routes after internalization (Bleil and Bretscher, 1982
; Iacopetta and Morgan, 1983
; Roberts et al., 2004
). Segregation of cargoes has been reported before: the EGF and Tfn receptors also sort to different CCP populations (Leonard et al., 2008
), and different GPCRs (G-protein coupled receptors) localize to distinct CCP subsets (Cao et al., 1998
; Mundell et al., 2006
; Puthenveedu and von Zastrow, 2006
). However, many other receptors are found in the same CCSs. For example, the LDL and Tfn receptors are predominantly found in the same CCSs (Keyel et al., 2006
). So some receptors enter through shared pits and some through different pits.
Even though integrin β1 and TfnR appear to internalize through different CCSs, both cargoes colocalize with both adaptors Dab2 and AP2 as well as EH domain proteins. These commonalities make it difficult to explain how the Dab2 NPF mutant inhibits internalization of integrin β1 but not of TfnR. One possibility is that integrin β1 internalization requires integrin β1–Dab2–EH domain complexes and TfnR internalization requires TfnR–AP2–EH domain complexes (). This model implies that cargo regulates EH domain protein function. Several studies suggest that cargo regulates internalization (Puthenveedu and von Zastrow, 2006
; Loerke et al., 2009
; Cao et al., 2010
; Liu et al.
, 2010; Mettlen et al., 2010
). Information on cargo occupancy may be relayed to the EH proteins by conformational changes in the adaptors. For example, AP2 undergoes conformation changes that enhance the affinity between AP2 and membranes after phosphorylation of AP2 by AAK1. In the new conformation, the YxxΦ sorting signal and PtdIns(4,5)P2
-binding sites are both exposed. The simultaneous binding of AP2 to multiple sites on the plasma membrane stabilizes the adaptor–membrane complex such that it can mediate clathrin assembly (Ricotta et al., 2002
; Honing et al., 2005
). In principle, cargo–adaptor complexes could then regulate the affinity of EH domain proteins for downstream accessory proteins, thus allowing for efficient internalization. Further experiments will be needed to understand whether and how EH domain protein function may be regulated by cargoes.
Why would adaptors need to bind both cargo and EH domain proteins for efficient endocytosis? One reason may be to ensure that only clathrin structures “fully loaded” with receptors are internalized. Clathrin pits with less than their full receptor capacity may not have enough functional EH domain proteins for internalization and may need to wait to recruit additional receptors. The lifetime of productive CCPs (not including plaques) ranges from 30 to >120 s (Loerke et al., 2009
). It is tempting to speculate that CCPs with longer lifetimes wait on the cell surface until they have their “full” receptor load and therefore sufficient functional EH domain proteins for internalization. Communication between receptors and EH domain proteins, mediated by endocytic adaptors like Dab2, may be important for regulating the endocytic checkpoint (Loerke et al., 2009
; Mettlen et al., 2009
). In the absence of Dab2 or in the absence of Dab2-associated EH domain proteins, the checkpoint may be incomplete and CCSs may escape past the checkpoint instead of being aborted, perhaps explaining the decrease in short-lifetime events (Supplemental Figure S4). However, there are other possible explanations, and more experiments will be required to determine the role of EH domain proteins in CCP dynamics and to fully understand the composition and regulation of the checkpoint.