Endocytosis of EGFR requires its kinase activity and N-terminal transmembrane dimerization motif.

EGFR signaling is attenuated by endocytosis and degradation of receptor-ligand complexes in lysosomes. Endocytosis of EGFR is known to be regulated by multiple post-translational modifications. The observation that prevention of these modifications does not block endocytosis completely, suggests the involvement of other mechanism(s). Recently, receptor clustering has been suggested to induce internalization of multiple types of membrane receptors. However, the mechanism of clustering-induced internalization remains unknown. We have used biparatopic antibody fragments from llama (VHHs) to induce EGFR clustering without stimulating tyrosine kinase activity. Using this approach, we have found an essential role for the N-terminal GG4-like dimerization motif in the transmembrane domain (TMD) for clustering-induced internalization. Moreover, conventional EGF-induced receptor internalization depends exclusively on this TMD dimerization and kinase activity. Mutations in this dimerization motif eventually lead to reduced EGFR degradation and sustained signaling. We propose a novel role for the TMD dimerization motif in the negative-feedback control of EGFR. The widely conserved nature of GG4-like dimerization motifs in transmembrane proteins suggests a general role for these motifs in clustering-induced internalization.

The munc13-4-rab27 complex is specifically required for tethering secretory lysosomes at the plasma membrane.

Cytotoxic T lymphocytes (CTLs) kill target cells through the polarized release of lytic molecules from secretory lysosomes. Loss of munc13-4 function inhibits this process and causes familial hemophagocytic lymphohistiocytosis type 3 (FHL3). munc13-4 binds rab27a, but the necessity of the complex remains enigmatic, because studies in knockout models suggest separate functions. In the present study, we describe a noncanonical rab27a-binding motif in the N-terminus of munc13-4. Point mutants in this sequence have severely impaired rab27a binding, allowing dissection of rab27a requirements in munc13-4 function. The munc13-4-rab27a complex is not needed for secretory lysosome maturation, as shown by complementation in CTLs from FHL3 patients and in a mast cell line silenced for munc13-4. In contrast, fusion of secretory lysosomes with, and content release at the plasma membrane during degranulation, strictly required the munc13-4-rab27a complex. Total internal reflection fluorescence microscopy imaging revealed that the complex corrals motile secretory lysosomes beneath the plasma membrane during degranulation and controls their docking. The propensity to stall motility of secretory lysosomes is lost in cells expressing munc13-4 point mutants that do not bind rab27. In summary, these results uncovered a mechanism for tethering secretory lysosomes to the plasma membrane that is essential for degranulation in immune cells.

A regulated interaction with the UIM protein Eps15 implicates parkin in EGF receptor trafficking and PI(3)K-Akt signalling.

Mutations in the parkin gene are responsible for a common familial form of Parkinson's disease. As parkin encodes an E3 ubiquitin ligase, defects in proteasome-mediated protein degradation are believed to have a central role in the pathogenesis of Parkinson's disease. Here, we report a novel role for parkin in a proteasome-independent ubiquitination pathway. We have identified a regulated interaction between parkin and Eps15, an adaptor protein that is involved in epidermal growth factor (EGF) receptor (EGFR) endocytosis and trafficking. Treatment of cells with EGF stimulates parkin binding to both Eps15 and the EGFR and promotes parkin-mediated ubiquitination of Eps15. Binding of the parkin ubiquitin-like (Ubl) domain to the Eps15 ubiquitin-interacting motifs (UIMs) is required for parkin-mediated Eps15 ubiquitination. Furthermore, EGFR endocytosis and degradation are accelerated in parkin-deficient cells, and EGFR signalling via the phosphoinositide 3-kinase (PI(3)K)-Akt pathway is reduced in parkin knockout mouse brain. We propose that by ubiquitinating Eps15, parkin interferes with the ability of the Eps15 UIMs to bind ubiquitinated EGFR, thereby delaying EGFR internalization and degradation, and promoting PI(3)K-Akt signalling. Considering the role of Akt in neuronal survival, our results have broad new implications for understanding the pathogenesis of Parkinson's disease.

Ligand-induced EGF receptor oligomerization is kinase-dependent and enhances internalization.

The current activation model of the EGF receptor (EGFR) predicts that binding of EGF results in dimerization and oligomerization of the EGFR, leading to the allosteric activation of the intracellular tyrosine kinase. Little is known about the regulatory mechanism of receptor oligomerization. In this study, we have employed FRET between identical fluorophores (homo-FRET) to monitor the dimerization and oligomerization state of the EGFR before and after receptor activation. Our data show that, in the absence of ligand, ∼40% of the EGFR molecules were present as inactive dimers or predimers. The monomer/predimer ratio was not affected by deletion of the intracellular domain. Ligand binding induced the formation of receptor oligomers, which were found in both the plasma membrane and intracellular structures. Ligand-induced oligomerization required tyrosine kinase activity and nine different tyrosine kinase substrate residues. This indicates that the binding of signaling molecules to activated EGFRs results in EGFR oligomerization. Induction of EGFR predimers or pre-oligomers using the EGFR fused to the FK506-binding protein did not affect signaling but was found to enhance EGF-induced receptor internalization. Our data show that EGFR oligomerization is the result of EGFR signaling and enhances EGFR internalization.

Homo-FRET imaging as a tool to quantify protein and lipid clustering.

Homo-FRET, Förster resonance energy transfer between identical fluorophores, can be conveniently measured by observing its effect on the fluorescence anisotropy. This review aims to summarize the possibilities of fluorescence anisotropy imaging techniques to investigate clustering of identical proteins and lipids. Homo-FRET imaging has the ability to determine distances between fluorophores. In addition it can be employed to quantify cluster sizes as well as cluster size distributions. The interpretation of homo-FRET signals is complicated by the fact that both the mutual orientations of the fluorophores and the number of fluorophores per cluster affect the fluorescence anisotropy in a similar way. The properties of the fluorescence probes are very important. Taking these properties into account is critical for the correct interpretation of homo-FRET signals in protein- and lipid-clustering studies. This is be exemplified by studies on the clustering of the lipid raft markers GPI and K-ras, as well as for EGF receptor clustering in the plasma membrane.

High cytotoxicity of cisplatin nanocapsules in ovarian carcinoma cells depends on uptake by caveolae-mediated endocytosis.

PURPOSE: Cisplatin nanocapsules, nanoprecipitates of cisplatin encapsulated in phospholipid bilayers, exhibit increased in vitro toxicity compared with the free drug toward a panel of human ovarian carcinoma cell lines. To elucidate the mechanism of cell killing by nanocapsules and to understand the cell line dependence of nanocapsule efficacy, the route of uptake and the intracellular fate of the nanocapsules were investigated. EXPERIMENTAL DESIGN: Intracellular platinum accumulation and cisplatin-DNA-adduct formation were measured in cell lines that differ in sensitivity to cisplatin nanocapsules. Confocal fluorescence microscopy in combination with down-regulation with small interfering RNA was used to map the route of cellular uptake of nanocapsules containing fluorescein-labeled cisplatin. RESULTS: In sensitive cell lines, cisplatin from nanocapsules is taken up much more efficiently than the free compound. In IGROV-1 cells, the increased platinum accumulation results in augmented cisplatin-DNA-adduct formation. Confocal fluorescence microscopy revealed that the uptake of nanocapsules is energy dependent. Colocalization with markers of early and late endosomes indicated uptake via endocytosis. Down-regulation of caveolin-1 with small interfering RNA inhibited the uptake and cytotoxic effect of nanocapsules in IGROV-1 cells. Ovarian carcinoma cells, in which the nanocapsules are less effective than in IGROV-1 cells, do not internalize the nanocapsules (OVCAR-3) or accumulate them in an endocytic compartment after clathrin-mediated endocytosis (A2780). CONCLUSIONS: The high cytotoxicity of cisplatin nanocapsules requires caveolin-1-dependent endocytosis that is followed by release of the drug from a late endosomal/lysosomal compartment and cisplatin-DNA-adduct formation. The findings may be applied in predicting the efficacy of nanoparticulate anticancer drug delivery systems in treating different tumor types.

Homo-FRET imaging enables quantification of protein cluster sizes with subcellular resolution.

Fluorescence-anisotropy-based homo-FRET detection methods can be employed to study clustering of identical proteins in cells. Here, the potential of fluorescence anisotropy microscopy for the quantitative imaging of protein clusters with subcellular resolution is investigated. Steady-state and time-resolved anisotropy detection and both one- and two-photon excitation methods are compared. The methods are evaluated on cells expressing green fluorescent protein (GFP) constructs that contain one or two FK506-binding proteins. This makes it possible to control dimerization and oligomerization of the constructs and yields the experimental relation between anisotropy and cluster size. The results show that, independent of the experimental method, the commonly made assumption of complete depolarization after a single energy transfer step is not valid here. This is due to a nonrandom relative orientation of the fluorescent proteins. Our experiments show that this relative orientation is restricted by interactions between the GFP barrels. We describe how the experimental relation between anisotropy and cluster size can be employed in quantitative cluster size imaging experiments of other GFP fusions. Experiments on glycosylphosphatidylinisotol (GPI)-anchored proteins reveal that GPI forms clusters with an average size of more than two subunits. For epidermal growth factor receptor (EGFR), we observe that approximately 40% of the unstimulated receptors are present in the plasma membrane as preexisting dimers. Both examples reveal subcellular heterogeneities in cluster size and distribution.

Membrane rearrangements mediated by coronavirus nonstructural proteins 3 and 4.

Coronaviruses replicate their genomes in association with rearranged cellular membranes. The coronavirus nonstructural integral membrane proteins (nsps) 3, 4 and 6, are key players in the formation of the rearranged membranes. Previously, we demonstrated that nsp3 and nsp4 interact and that their co-expression results in the relocalization of these proteins from the endoplasmic reticulum (ER) into discrete perinuclear foci. We now show that these foci correspond to areas of rearranged ER-derived membranes, which display increased membrane curvature. These structures, which were able to recruit other nsps, were only detected when nsp3 and nsp4 were derived from the same coronavirus species. We propose, based on the analysis of a large number of nsp3 and nsp4 mutants, that interaction between the large luminal loops of these proteins drives the formation of membrane rearrangements, onto which the coronavirus replication-transcription complexes assemble in infected cells.

EGF induces coalescence of different lipid rafts.

The suggestion that microdomains may function as signaling platforms arose from the presence of growth factor receptors, such as the EGFR, in biochemically isolated lipid raft fractions. To investigate the role of EGFR activation in the organization of lipid rafts we have performed FLIM analyses using putative lipid raft markers such as ganglioside GM1 and glycosylphosphatidylinositol (GPI)-anchored GFP (GPI-GFP). The EGFR was labeled using single domain antibodies from Llama glama that specifically bind the EGFR without stimulating its kinase activity. Our FLIM analyses demonstrate a cholesterol-independent colocalization of GM1 with EGFR, which was not observed for the transferrin receptor. By contrast, a cholesterol-dependent colocalization was observed for GM1 with GPI-GFP. In the resting state no colocalization was observed between EGFR and GPI-GFP, but stimulation of the cell with EGF resulted in the colocalization at the nanoscale level of EGFR and GPI-GFP. Moreover, EGF induced the enrichment of GPI-GFP in a detergent-free lipid raft fraction. Our results suggest that EGF induces the coalescence of the two types of GM1-containing microdomains that might lead to the formation of signaling platforms.

Ubiquilin recruits Eps15 into ubiquitin-rich cytoplasmic aggregates via a UIM-UBL interaction.

Eps15 and its related protein Eps15R are key components of the clathrin-mediated endocytic pathway. We searched for new binding partners of Eps15 using a yeast two-hybrid screen. We report here that ubiquilin (hPLIC1), a type-2 ubiquitin-like protein containing a ubiquitin-like domain (UBL) and a ubiquitin-associated domain (UBA), interacts with both Eps15 and Eps15R. Using glutathione-S-transferase pull-down experiments, we show that the first ubiquitin-interacting motif of Eps15 (UIM1) interacts directly with the UBL domain of ubiquilin, whereas it does not bind to ubiquitinated proteins. The second UIM of Eps15 (UIM2) binds poorly to the UBL domain but does bind to ubiquitinated proteins. Two other UIM-containing endocytic proteins, Hrs and Hbp, also interact with ubiquilin in a UIM-dependent manner, whereas epsin does not. Immunofluorescence analysis showed that endogenous Eps15 and Hrs, but not epsin, colocalize with green-fluorescent-protein-fused ubiquilin in cytoplasmic aggregates that are not endocytic compartments. We have characterized these green-fluorescent-protein-fused-ubiquilin aggregates as ubiquitin-rich intracytoplasmic inclusions that are recruited to aggresomes upon proteasome inhibition. Moreover, we show that endogenous Eps15 and endogenous ubiquilin colocalize to cytoplasmic aggregates and aggresomes. Finally, we show that the recruitment of Eps15 into ubiquilin-positive aggregates is UIM dependent. Altogether, our data identify ubiquilin as the first common UIM-binding partner of a subset of UIM-containing endocytic proteins. We propose that this UIM/UBL-based interaction is responsible for the sequestration of certain UIM-containing endocytic proteins into cytoplasmic ubiquitin-rich protein aggregates.

Sorting of ligand-activated epidermal growth factor receptor to lysosomes requires its actin-binding domain.

Ligand-induced down-regulation of the epidermal growth factor receptor (EGFR) comprises activation of two sequential transport steps. The first involves endocytic uptake by clathrin-coated vesicles, the second transfer of endocytosed EGFR from endosomes to lysosomes. Here we demonstrate that the second transport step requires a domain of the EGFR that encompasses residues 985-996 and was previously found to interact with actin. Deletion of domain 989-994 (Delta989-994 EGFR) did not interfere with EGFR uptake but completely abrogated its degradation. In contrast, both uptake and degradation were affected for K721A EGFR, a kinase-deficient EGFR mutant. To measure intracellular EGFR sorting, we developed a novel cell fractionation assay toward which cells were co-transfected for chicken hepatic lectin, a receptor for agialoglycoproteins. These cells were incubated with agialofetuin-coupled colloidal gold, which was targeted to lysosomes after receptor-mediated endocytosis. Compartments within the lysosomal pathway gained buoyant density because of the presence of colloidal gold and could be isolated from cell homogenates by ultracentrifugation through a high-density sucrose cushion. In contrast to endocytosed wild type EGFR, both Delta989-994 EGFR and K721A EGFR were largely not retrieved in gold-containing endocytic compartments. These results are supported with morphological data. We conclude that sorting of endocytosed EGFR into the degradation pathway requires both its kinase activity and actin-binding domain.