Clathrin-independent endocytosis of ubiquitinated cargos

Abstract

Plasma membrane receptors can be endocytosed through clathrindependent and clathrin-independent pathways. Here, we show that the epidermal growth factor (EGF) receptor (EGFR), when stimulated with low doses of EGF, is internalized almost exclusively through the clathrin pathway, and it is not ubiquitinated. At higher concentrations of ligand, however, a substantial fraction of the receptor is endocytosed through a clathrin-independent, lipid raft-dependent route, as the receptor becomes ubiquitinated. An ubiquitination-impaired EGFR mutant was internalized through the clathrin pathway, whereas an EGFR͞ubiquitin chimera, that can signal solely through its ubiquitin (Ub) moiety, was internalized exclusively by the non-clathrin pathway. Non-clathrin internalization of ubiquitinated EGFR depends on its interaction with proteins harboring the Ub-interacting motif, as shown through the ablation of three Ub-interacting motif-containing proteins, eps15, eps15R, and epsin. Thus, eps15s and epsin perform an important function in coupling ubiquitinated cargo to clathrin-independent internalization.internalization ͉ rafts ͉ caveolae ͉ ubiquitination ͉ ubiquitin-interacting motif U biquitination is a posttranslational modification whereby substrate proteins are conjugated to a short highly conserved peptide, ubiquitin (Ub), through the action of Ub ligases (E3 enzymes). Polyubiquitination, in which a chain of Ub is appended, targets proteins to proteasomal degradation (1). However, when a single Ub moiety is appended (monoubiquitination), the modification functions as a signaling device through interactions with intracellular proteins harboring Ub-binding domains, such as the Ub-interacting motif (UIM) (2). In yeast, monoubiquitination has been known to act as an internalization signal for quite some time (3). In mammals, however, this connection has remained more elusive.We are interested in the mechanisms of internalization of receptor tyrosine kinases and, in particular, the epidermal growth factor receptor (EGFR). The EGFR is monoubiquitinated at multiple sites (4) through the action of the E3 enzyme Cbl. Although there is consensus on the function of Cbl and receptor ubiquitination in intracellular sorting of the EGFR, their role in the internalization step of endocytosis is less clear (5, 6). To gain insight into this issue, we generated a chimera in which the extracellular and transmembrane domains of the EGFR are fused to a mutant Ub (Ubmut), unable to form polyUb chains (EGFR͞Ubmut). With this chimera, we showed that ubiquitination is sufficient for internalization (4). The present studies were undertaken to elucidate the molecular mechanisms through which receptor ubiquitination directs internalization. Materials and MethodsTransfection and Biochemical Studies. Transfections were performed by using Lipofectamine or Oligofectamine (Invitrogen). For biochemical experiments, cells were serum-starved and then stimulated with EGF (100 ng͞ml, unless otherwise indicated) at 37°C. Lysis, immunoprecipitation, and immunoblot...

Distinctive activation patterns in constitutively active and gefitinib-sensitive EGFR mutants

“…Regardless of their phosphorylation and ubiquitination properties, we found no apparent difference in receptor internalization among wild-type and mutant EGFRs (Supplementary Figure 2). This result is consistent with previous reports showing that EGFR ubiquitination is not essential for receptor internalization (Duan et al, 2003;Jiang and Sorkin, 2003;Sigismund et al, 2005). However, S768I, L861Q, E709G, and G719S mutants did have more sustained Tyr phosphorylation than wild-type receptor in response to EGF (Figure 7A), suggesting a defect(s) in the negatively regulatory mechanism.…”

“…The role of EGFR phosphorylation at Tyr 1045 in ligand-induced receptor ubiquitination, endocytosis, and degradation was demonstrated previously (Levkowitz et al, 1999;Mosesson et al, 2003). Although the necessity of Tyr 1045 phosphorylation and receptor ubiquitination in EGFR internalization has been questioned in several studies (Duan et al, 2003;Jiang and Sorkin, 2003;Sigismund et al, 2005), our data showed that S768I, L861Q, and E709G mutants had very high levels of Tyr 1045 phosphorylation (Figures 4, 6, and 7). Mutant G719S had lower EGF-induced phosphorylation at other Tyr residues, but had stronger phosphorylation at Tyr 1045, than wild-type receptor (Figures 4 and 6).…”

Raf plus TGFβ-dependent EMT is initiated by endocytosis and lysosomal degradation of E-cadherin

“…Nystatin that blocks the caveolin-dependent endocytosis reduces the number of round E-cadherin vesicles and some caveolin-positive E-cadherin vesicles could occasionally be detected. E-cadherin may well be endocytosed by both pathways under certain conditions, similarly to EGF receptors as reported recently (Sigismund et al, 2005). Further studies would be required to validate the relationship between clathrin-and caveole-mediated endocytosis of adherens junction proteins in polarized/ depolarized epithelial cells vs cells undergoing EMT.…”

Transactivation of the epidermal growth factor receptor in responses to myocardial stress and cardioprotection

“…dissociation of EGFR signal elements from caveolar domains), internalisation of raft-localised EGFR may thus be fundamental to signal transduction (Balbis and Posner, 2010). Data collectively support activity-dependent EGFR internalisation, with low-level agonism triggering clathrin-dependent/cholesterolindependent recycling to the cell membrane (Sigismund et al, 2005;Sigismund et al, 2008), whereas higher level agonism results in clathrin-independent/cholesteroldependent EGFR internalisation and trafficking to late endosomes (Lai et al, 1989).…”