The endocytic pathway followed by RTKs, and in particular by EGFR, has been widely studied (reviewed in Sorkin and Waters, 1993
). Upon ligand binding, EGFR enters clathrin-coated pits at the cell surfaces, is internalized by clathrin-coated vesicles, and reaches early endosomes where sorting occurs to the plasma membrane for receptor recycling or to late endosomes and lysosomes for down-regulation. Several lines of evidence (reviewed in Di Fiore et al., 1997
) suggest that eps15 plays a role in the control of endocytosis, a possibility that is antagonized, however, by the observation that eps15 distribution does not change appreciably upon ligand-induced endocytosis of the EGFR (Tebar et al., 1996
; Van Delft et al., 1997b
). We were conversely able to show that the eps15 undergoes dramatic relocalization during EGFR endocytosis, by first being recruited to the plasma membrane and then colocalizing with the EGFR in early and late endosomes.
In our opinion, the initial relocalization of eps15 to the plasma membrane deserves particular attention. Two major models for RTK-mediated internalization have been proposed. In the first, ligand-bound receptors enter coated pits simply by virtue of their random lateral mobility in the plasma membrane. Once they reach the pit, they are retained by specific components of the pit machinery. The best candidate for such a role is the clathrin adaptor complex protein AP-2. In the EGFR system, AP-2 binds in a ligand-dependent manner (Sorkin and Carpenter, 1993
) to a tyrosine-based signal, which is part of one of the three endocytic codes present in the C-terminal portion of the EGFR. Binding of AP-2 to the EGFR requires activation of the receptor kinase activity, autophosphorylation, and conformational changes that probably unmask the binding site; however, endocytosis of EGFR is a second-order saturable process, likely involving competition among EGFRs for a downstream component of the endocytic machinery (Lund et al., 1990
). Quantitative considerations (reviewed in Nesterov et al., 1995b
) militate against AP-2 being such a component. In addition, AP-2-binding mutants of the EGFR display unperturbed internalization kinetics (Nesterov et al., 1995b
), at least at low levels of receptor expression (Sorkin et al., 1996
). In addition, evidence has been provided that a tyrosine kinase substrate, different from EGFR itself, is needed for efficient recruitment into coated pits of ligand-activated EGFR (Lamaze and Schmid, 1995
In a second model, a membrane-bound docking apparatus (or several receptor-specific apparatuses) might exist that facilitates targeting of receptors to coated pits. The docking molecule (or a component of the docking apparatus) might coincide with the aforementioned tyrosine kinase substrate, other than the EGFR. Eps15 possesses several of the characteristics that fit this role: 1) it is an EGFR substrate (Fazioli et al., 1993
); 2) it is an essential component of the endocytic pathway (Carbone et al., 1997
); 3) it localizes to the plasma membrane and colocalizes with the EGFR after ligand stimulation (this study); and 4) it is constitutively bound to AP-2 (Benmerah et al., 1995
). One might thus postulate that constitutive nonplasma membrane-bound eps15/AP-2 complexes are recruited to the plasma membrane by EGFR-induced posttranslational modifications of eps15. Tyrosine phosphorylation is the most obvious candidate for such a modification, albeit other modifications, such as the recently shown monoubiquitination of eps15 (Van Delft et al., 1997a
), cannot be excluded at this stage. The relocalization of AP-2 to the plasma membrane can then trigger assembly of the clathrin lattice and receptor retention into the pit through various mechanisms (reviewed in Kirchhausen et al., 1997
). At this stage eps15 might dissociate from AP-2, as indicated by recent findings of Cupers et al. (1998)
The above model would predict that plasma membrane relocalization and tyrosine phosphorylation of eps15 are independent of binding of AP-2 to the receptor: a prediction that was experimentally validated in this study. In addition, this model would easily accommodate experimental findings such as the preferential localization of eps15 at the rims of the coated pits (Tebar et al., 1996
). As originally pointed out by Tebar et al. (1996)
, the localization of eps15 at the rims of coated pits is surprising, because AP-2, the binding partner, is localized throughout the coat profile. This observation led Kirchhausen et al. (1997)
to postulate that eps15 might undergo cycles of binding to and release from AP-2 during coat assembly. In our proposed model, the location of eps15 at the rim, which is likely the growing part of a forming pit, would be self-explanatory, if eps15 were to function as a docking/recruiting molecule.
Recruitment of eps15 to the plasma membrane does not require a stable detergent-resistant physical interaction (direct or indirect) between eps15 and the EGFR (this study). In addition, a stoichiometrically significant interaction between eps15 and EGFR could not be immediately reconciled with the absence of eps15 from the deeper parts of coated pits (Tebar et al., 1996
) where EGFR and AP-2 are abundant. Indeed, there is discordance in the literature as to whether eps15 is complexed to the EGFR in vivo (Fazioli et al., 1993
; Van Delft et al., 1997b
; this study). Part of the differences can be cell-specific because in a survey of several cell lines expressing the EGFR we identified some cell lines in which eps15 and EGFR can indeed be coimmunoprecipitated (our unpublished results), albeit with rather low stoichiometry: <1% of the eps15 pool. The lack of universality of the phenomenon and its quantitative limitedness appear to militate against its relevance; however, one can envision a transitory, unstable interaction between eps15 and EGFR at the recruiting edge of the pit, which might account for all of the above observations.
An interesting finding is that in addition to the expected association with coated pits at the cell surface, eps15 is localized on intracellular vesicles not only peripheral, but also perinuclear. EGFR activation and endocytosis determines first a recruitment of eps15 at the cell surface and later a redistribution toward the central area of the cell. This phenomenon, although not involving the total eps15 pool, clearly reflects the behavior of a large amount of eps15 molecules. In addition, eps15 colocalizes with the EGFR during endocytosis. Thus, our findings pose the question as to whether eps15 is “trafficked” with the EGFR from the plasma membrane to the endosomes or whether it is “targeted” to endosomal membranes. We note that although we were able to readily detect association of eps15 with coated pits and early and late endosomes, we could not detect its presence in clathrin-coated vesicles. This result is consistent with those reported by Cupers et al. (1998)
, who showed that purified coated vesicles contain negligible amounts of eps15 and that eps15 is lost during coat assembly in vitro. Thus, we favor the possibility that multiple cycles of association:dissociation of eps15 with membranes occur as the EGFR moves forward in the endocytic pathway. The molecular determinants involved in the “retargeting” of eps15 to endosomes are unknown; however, the kinetics of eps15 tyrosine phosphorylation correlates with its reassociation to endosomes. A late peak of eps15 tyrosine phosphorylation occurs, in fact, in a nocodazole-sensitive compartment. Thus, in analogy with what we propose for eps15 recruitment to the plasma membrane, the EGFR might be able to phosphorylate eps15 in the endosomal compartment, thereby determining its association with endosomal membranes.
Also somewhat surprising is our finding that eps15 colocalizes with α-adaptin not only at the plasma membrane, in cells treated with EGF at 4°C, but also in perinuclear structures after warming at 37°C. We do not know whether these structures also contain internalized EGFR, thus representing bona fide early or late endosomes. On the basis of current knowledge, this possibility appears unlikely. These structures, however, might represent intermediate carrier vesicles, other than endosomes, involved in the receptor transport and/or sorting along the endocytic pathway.
The retargeting of eps15 to endosomes, after its dissociation from coated vesicles, strongly suggests that eps15 plays a role in the late steps of endocytosis, different from that exerted at the plasma membrane level in the formation of coated pits. Indeed some molecular evidence already exists that these two putative functions of eps15 can be dissociated. We have shown previously that microinjection of anti-eps15 antibodies efficiently inhibits internalization of EGFR (Carbone et al., 1997
). In addition, overexpression of a dominant negative mutant comprising only the AP-2 binding region of eps15 (L2 region) inhibited internalization of EGFR and establishment of successful infection by Sindbis virus, which is known to require coated pits-mediated internalization of the virus (reviewed in Marsh and Helenius, 1989
). Surprisingly, an eps15 mutant encompassing only its three EH domains had no effect on EGFR internalization but completely inhibited Sindbis infection (Carbone et al., 1997
; our unpublished observations). One possible explanation for this discrepancy is that the inhibition by EH domains is exerted at a step in the endocytic pathway other than internalization, as for example in the transport from early endosomes to the more acidic late endosomes, a step required for viral fusion and infection (Marsh and Helenius, 1989
). If this were true, it would direct the attention regarding a possible role of eps15 in late endocytosis to the protein:protein interaction abilities of the EH domain, an intriguing possibility that warrants further investigation.