By analogy with the recruitment of signaling molecules to activated growth factor receptors (Figure 3), it has been suggested that the activation of signaling receptors could also recruit the molecular machinery for de novo CCP assembly for their own internalization. The de novo formation model was widely seen as a valid model reflecting the ligand-induced internalization of EGF-Rs and GPCRs. However, using another receptor model for ligand-induced endocytosis (high-affinity immunoglobulin E receptor), Santini et al. provided evidence that CCPs are not assembled at the site of receptor activation and challenged the de novo assembly model for signaling receptors (84). An alternative model would be that activated receptors are targeted to CCPs either already formed or in the process of assembly before receptor activation (preexisting CCPs). These two models, which remain a matter of debate, involve major differences in CCP behavior. De novo CCP assembly implies that receptor activation regulates the CCP assembly machinery and that newly formed CCPs are probably restricted to the stimulated receptor. The preexisting model implies that receptor activation controls cargo recruitment to CCPs but not CCP assembly, and suggests that activated receptors share CCPs with cargos taken up by constitutive endocytosis.
GPCRs, targeting to preexisting CCPs?
The de novo assembly model was put forward following studies of the endocytosis of GPCRs. In this particular case, GPCR internalization requires additional molecules – βarrs (βarr1 and βarr2) – that were initially identified as proteins required for desensitization of the β-adrenergic receptor. βarrs bind to GPCRs upon agonist activation, and interact directly with both clathrin and AP-2 (85–87). These findings raised the possibility that GPCR/βarr complexes could drive the assembly of their own CCPs in response to receptor activation (Figure 3). However, live-cell imaging studies combining different GPCRs (thyrotropin-releasing hormone, β2-adrenergic and dopamine receptors), with either βarr1 or βarr2, and various markers for CCPs (Eps15 or clathrin light chain) clearly showed that GPCR/βarr complexes were recruited to CCPs assembled before agonist stimulation (88–90).
It remains possible that the preexisting CCPs to which activated GPCRs are recruited differ from other CCPs. If this were the case, only some of the preexisting CCPs would be competent for the recruitment of activated GPCRs. Initial studies in live and fixed cells overexpressing both GPCRs and βarrs showed that βarr/GPCR complexes were recruited to almost all of the CCPs present at the cell surface [(88–90) and unpublished observations] and that internalizing GPCR were found into structures that were positive for Tf/Tf-R (89,91). Interestingly, Puthenveedu and Von Zastrow, using a similar approach, found that the proportion of CCPs containing activated GPCR decreased to 50% if only GPCR was overexpressed (90), consistent with the results of another recent study (92). These results suggest that GPCR/βarr complexes are targeted to specific subsets of preexisting CCPs. However, the observation that the proportion of CCPs containing activated GPCR increases with βarr levels suggests that GPCR/βarr complexes can be recruited to all preexisting CCPs, the extent of this recruitment being limited by the amount of cytoplasmic βarr available. This is consistent with the requirement for binding to both AP-2 and clathrin for the targeting of βarrs to CCPs (93,94), and with the fact that AP-2 and clathrin binding only could not provide the information for targeting to a specific subset of CCPs. As expected, mutated forms of βarrs, which mimic activation by agonist, are found in most if not all CCPs at steady state (94), indicating that βarrs are unlikely to represent a targeting device for specific CCPs.
EGF-R, the de novo CCP cargo prototype?
Most of the support in favor of the de novo model of CCP formation comes from studies on endocytosis of the EGF-R. Unoccupied EGF-Rs are not efficiently internalized, whereas EGF-Rs are rapidly taken up into CCPs after activation. Indeed, after much debate (95,96), it was finally established that the tyrosine kinase activity of the EGF-R is required for the recruitment of EGF-R/EGF complexes into CCPs (97). Furthermore, the EGF-R is one of the very few receptors that can be coimmunoprecipitated with the AP-2 complex (98). The occurrence of this interaction upon EGF activation has been seen as strong evidence for EGF-driven de novo assembly of EGF-specific CCPs through the specific recruitment of AP-2 to activated EGF-R (Figure 3).
This hypothesis was challenged by the observation that mutated EGF-R unable to bind to AP-2 was nonetheless internalized (99). Similarly, cells depleted of AP-2 or expressing a mutated form of the μ2 subunit unable to bind activated EGF-R have been shown to internalize EGF (21,100), suggesting that CCPs devoid of functional AP-2 remain competent for EGF-R endocytosis. The apparent dependence of EGF-R internalization on clathrin under the same conditions (21,100), suggests that activated EGF-R may be internalized through AP-2-independent CCPs, like the LDL-R. However, it has also been reported that EGF internalization may be affected by AP-2 depletion (52,101). These apparent discrepancies may be because of differences in the experimental conditions used in these studies. Indeed, factors such as cell type, EGF concentration and stimulation conditions may determine the endocytic pathway taken by activated EGF-R (102–106). Thus, clathrin- and AP-2-dependent internalization of the EGF-R occurs when the receptor is directly stimulated by a low dose of EGF (∼1–3 ng/mL), at 37°C, in cells not starved of serum (52). Under these conditions, EGF internalization is inhibited or delayed by the depletion of endogenous AP-2, clathrin heavy chain or UIM-containing proteins, including Eps15/Eps15R and epsins (52,101,105).
Other reports have suggested that EGF treatment induces the assembly of EGF-specific CCPs. An initial study by Brodsky et al. reported that at high doses of EGF (250 ng/mL), with prebinding at 4°C, in serum-starved cells, rapid tyrosine phosphorylation of the clathrin heavy chain occurred in a c-src-dependent manner, resulting in the massive recruitment of clathrin to the plasma membrane, and correlated with an increase in EGF uptake (107). These observations suggest that, under these conditions, activated EGF-R can drive the assembly of new CCPs. This hypothesis was further confirmed by an electron microscopy analysis, which showed that the number of CCPs doubled in cells stimulated by EGF at 4°C. These CCPs contained EGF-R, AP-2 and clathrin, together with EGF-induced signaling molecules (108). It has also recently been shown that EGF-R activation increases the activity of the phosphatidylinositol kinase type I, which produces PI(4,5)P2, the phosphoinositide specifically involved in AP-2 recruitment at the plasma membrane (109). These results suggest an alternative mechanism through which EGF treatment may activate the molecular machinery required for the de novo assembly of AP-2-containing CCPs. The EGF-driven assembly of CCPs has also been observed in cells depleted of AP-2 (101). This clearly indicates again that CCP can be formed in the absence of AP-2, however, it does not demonstrate that this process occurs in ordinary conditions.
Thus, the incubation of cells with EGF at 4 °C can induce the assembly of new CCPs, in a process that may be independent of the AP-2 complex. In addition, EGF-induced CCPs appear to be specific to the EGF-R, or at least they contain fewer Tf-R than the ‘classical’ CCPs found in unstimulated cells (101,108) and contain markers of rafts (GM1) normally excluded from Tf-R-containing CCPs (108), suggesting that they are different from the CCPs involved in constitutive endocytosis. These recent studies are consistent with previous findings that the molecular machineries involved in the recruitment of Tf and EGF receptors to CCPs differ (46). They are also consistent with the convincing demonstrations that EGF-R does not compete with Tf-R for endocytosis, regardless of the EGF concentration used or the number of EGF-R expressed at the cell surface (103,104,110). Similarly, dileucine and tyrosine-based signals do not compete with each other for internalization. The lack of competition between Tf-R and EGF-R for endocytosis implies only that the two receptors do not compete for the same sorting device – the μ2 subunit of AP-2. Finally, the absence of a positive effect of EGF stimulation on Tf endocytosis (103,104), despite the induction of new CCP formation, suggests that the newly formed CCPs cannot sort Tf-R. This is consistent with electron microscopy data (see above) and the absence of TTP in EGF-R-positive CCPs (55).
As AP-2 can be dispensable for EGF-R clathrin-dependent endocytosis, alternative adaptors or a specific molecular machinery may exist for the recruitment of activated EGF-R, as described above for LDL-R or ubiquitin. Several proteins are good candidates for this role. Perforated cell assays have shown that a substrate of the EGF-R tyrosine kinase is required for the efficient recruitment of activated EGF-R, but not of Tf-R, into CCPs (97). This substrate may be Eps15, as it was later shown that the tyrosine phosphorylation of Eps15 was required for efficient targeting of EGF-R, but not of Tf-R to CCPs (111). Eps15 does bind AP-2 (112), but is unlikely to substitute for AP-2 in the assembly of CCPs, as it cannot interact with clathrin (113), and its incorporation into CCPs is dependent on AP-2 (33,82). Interestingly, downregulation of CALM, the ubiquitous form of neuronal AP180, has been shown to inhibit the endocytosis of EGF-R, but not that of Tf (52), suggesting that this protein plays a specific role in EGF-R endocytosis. CALM is found in CCPs at steady state (114), and AP180 has been reported to be sufficient for clathrin assembly on a lipid monolayer (115). CALM is therefore a good candidate for involvement in the assembly of EGF-R-specific CCPs. However, further studies are required to understand the precise role of this molecule, as overexpression of CALM mutants has blocked Tf-R endocytosis (114).
The recruitment of EGF-R to CCPs also appears to require proteins previously identified as involved in signal transduction. For example, Grb2 has been shown to be required for the endocytosis of EGF (116) during the recruitment of activated EGF-R to CCPs (52,101,117). The precise mechanism of action of Grb2 remains unclear, but may involve the interaction of this molecule with the ubiquitin ligase Cbl (118,119). It remains unclear how this complex becomes linked to a clathrin assembly protein responsible for the final assembly of CCPs. It was initially suggested that the ubiquitination of EGF-R might be involved. However, neither the ubiquitin ligase activity of Cbl nor the ubiquitination of the EGF-R itself are required for CCP clathrin-dependent endocytosis of the EGF-R (118,120). Identification of the final adaptor for EFG-R would provide real insight into this complex internalization process.
Other signaling receptors possibly involved in de novo CCP assembly
A few other receptors can, like EGF-R, induce the assembly of their own endocytic structures. One example is the nerve growth factor receptor (NGF-R). Early studies indicated that the treatment of NGF-starved cells with NGF or EGF increased the total number of CCPs present at the plasma membrane (121,122). In this case, the activation of the NGF-R results in an increase in AP-2 and clathrin levels at the plasma membrane and an increase in clathrin heavy chain phosphorylation (123), as observed for EGF-R (see above). However, in contrast to what has been observed for EGF, NGF treatment also stimulates Tf internalization (123), suggesting that the NGF-induced CCPs do not differ from the CCPs involved in constitutive endocytosis.
An increase in clathrin recruitment at the plasma membrane and/or in tyrosine phosphorylation of the clathrin heavy chain has also been described for the insulin receptor (124,125) and for the B-cell and T-cell receptors [BCR and TCR, respectively; (126,127)]. Ligation of the BCR and TCR results in the tyrosine phosphorylation of clathrin and the endocytosis of these receptors through a clathrin-dependent internalization pathway. The BCR is internalized only if phosphorylated clathrin is associated with lipid rafts. The Tf-R was not found associated with these lipid rafts, suggesting that BCR endocytosis occurs through a different set of CCPs, as suggested for EGF-induced CCPs (108).
Thus, although it is clear that EGF treatment under specific conditions can induce the formation of CCPs that are specifically involved in the internalization of EGF-R, or at least not involved in the internalization of Tf, the existence of similar mechanisms for other receptors remains possible but unproven. Direct morphological studies and live-cell imaging would be useful and might make it possible to link clathrin modifications with changes in the CCP assembly process at the plasma membrane.
CCPs and pathogens
A number of viral pathogens enter cells by receptor-mediated endocytosis, targeting membrane receptors internalized by clathrin-dependent or clathrin-independent endocytosis (reviewed in 128). Two recent studies have investigated the dynamics of entry of the influenza virus (129) and reovirus (47) with respect to clathrin. In both studies, the virus was found to bind initially to the plasma membrane, on which it remained static. Clathrin staining was observed a few minutes later, at sites of virus attachment, followed by the endocytosis of the viral particles, as shown by the rapid disappearance of clathrin staining and rapid lateral movements of the internalized particles. In both cases, these events are consistent with the possibility that viruses may induce the assembly of their own clathrin-coated structures for internalization. These structures probably correspond to CCPs, as an early morphological study showed that influenza virus particles were present in CCPs and nascent vesicles (130).
A recent study unexpectedly showed that internalization of the bacterium Listeria monocytogenes was affected by depletion of the heavy chain of clathrin (131). Interestingly, an siRNA screen revealed that AP-2 was not involved, whereas dynamin, Eps15 and a number of signaling proteins, including Grb2, the met receptor and Cbl, were required for Listeria entry. These results suggest that Listeria may use the met receptor to induce the assembly of clathrin-coated structures similar to those induced by EGF-R stimulation (see above). However, bacteria are larger than classical CCPs, and electron microscopy studies are required to investigate this issue.