Fangtian Huang

Interaction of EGF receptor and Grb2 in living cells visualized by fluorescence resonance energy transfer (FRET) microscopy

The interaction of activated epidermal growth factor receptor (EGFR) with the Src homology 2 (SH2) domain of the growth-factor-receptor binding protein Grb2 initiates signaling through Ras and mitogen-activated protein kinase (MAP kinase) [1,2]. Activation of EGFRs by ligand also triggers rapid endocytosis of EGF-receptor complexes. To analyze the spatiotemporal regulation of EGFR-Grb2 interactions in living cells, we have combined imaging microscopy with a modified method of measuring fluorescence resonance energy transfer (FRET) on a pixel-by-pixel basis using EGFR fused to cyan fluorescent protein (CFP) and Grb2 fused to yellow fluorescent protein (YFP). Efficient energy transfer between CFP and YFP should only occur if CFP and YFP are less than 50A apart, which requires direct interaction of the EGFR and Grb2 fused to these fluorescent moieties [3]. Stimulation by EGF resulted in the recruitment of Grb2-YFP to cellular compartments that contained EGFR-CFP and a large increase in FRET signal amplitude. In particular, FRET measurements indicated that activated EGFR-CFP interacted with Grb2-YFP in membrane ruffles and endosomes. These results demonstrate that signaling via EGFRs can occur in the endosomal compartment. The work also highlights the potential of FRET microscopy in the study of subcellular compartmentalization of protein-protein interactions in living cells.

Multiple mechanisms collectively regulate clathrin-mediated endocytosis of the epidermal growth factor receptor.

Four independent mechanisms for uptake of activated EGFR are identified by a combination of receptor mutagenesis and RNA interference approaches.

EGF receptor ubiquitination is not necessary for its internalization

Ubiquitination of the EGF receptor (EGFR) has been implicated in EGF-induced receptor internalization, lysosomal degradation, and down-regulation. Mutation of EGFR ubiquitination sites identified by mass spectrometry yielded receptor mutants that are weakly ubiquitinated and not down-regulated by EGF. However, these EGFR mutants were normally internalized. To examine whether this internalization was mediated by the residual ubiquitination, systematic mutagenesis of lysine residues in the kinase domain of the EGFR was performed to generate a receptor mutant that is not ubiquitinated. Mutations of a number of lysines inhibited kinase activity of the EGFR, thus leading to the inhibition of receptor internalization. However, a mutant lacking 15 lysine residues (15KR), which was negligibly ubiquitinated and normally phosphorylated, was internalized at a rate similar to that of the wild-type EGFR. As in the case of the wild-type EGFR, internalization of the 15KR mutant depended on the presence of clathrin, Grb2 adaptor, and Cbl ubiquitin ligase. These data imply that EGFR ubiquitination is not necessary for its internalization by clathrin-coated pits. Interestingly, the reconstitution of two major ubiquitination sites in the 16KR receptor mutant, which had impaired kinase activity and slow internalization kinetics, resulted in a partial rescue of ubiquitination and a complete rescue of receptor internalization. This result suggests that ubiquitination of the kinase-impaired receptor can mediate its internalization by the clathrin pathway. Altogether these data emphasize the robustness of the EGFR internalization process, which can be controlled by multiple kinase- and ubiquitination-dependent and -independent mechanisms.

Lysine 63-linked polyubiquitination is required for EGF receptor degradation

Significance Many proteins are modified by the covalent attachment of a small polypeptide called ubiquitin to their lysine residues. Lysines in ubiquitin itself are further ubiquitinated, leading to formation of ubiquitin chains. Single ubiquitins and ubiquitin chains attached to integral membrane proteins, such as receptors, transporters, and channels, serve as molecular signals mediating endocytosis of these proteins and their subsequent targeting to lysosomes for degradation. This work uses quantitative mass spectrometry to show that activated EGF receptor is ubiquitinated by one to two short polyubiquitin chains linked via ubiquitin lysine 63 or conjugated with a single monoubiquitin. It is demonstrated that these Lys63-linked polyubiquitin chains are necessary for efficient targeting of EGF receptor to the lysosomal degradation pathway. Ubiquitination mediates endocytosis and endosomal sorting of various signaling receptors, transporters, and channels. However, the relative importance of mono- versus polyubiquitination and the role of specific types of polyubiquitin linkages in endocytic trafficking remain controversial. We used mass spectrometry-based targeted proteomics to show that activated epidermal growth factor receptor (EGFR) is ubiquitinated by one to two short (two to three ubiquitins) polyubiquitin chains mainly linked via lysine 63 (K63) or conjugated with a single monoubiquitin. Multimonoubiquitinated EGFR species were not found. To directly test whether K63 polyubiquitination is necessary for endocytosis and post-endocytic sorting of EGFR, a chimeric protein, in which the K63 linkage-specific deubiquitination enzyme AMSH [associated molecule with the Src homology 3 domain of signal transducing adaptor molecule (STAM)] was fused to the carboxyl terminus of EGFR, was generated. MS analysis of EGFR-AMSH ubiquitination demonstrated that the fraction of K63 linkages was substantially reduced, whereas relative amounts of monoubiquitin and K48 linkages increased, compared with that of wild-type EGFR. EGFR-AMSH was efficiently internalized into early endosomes, but, importantly, the rates of ligand-induced sorting to late endosomes and degradation of EGFR-AMSH were dramatically decreased. The slow degradation of EGFR-AMSH resulted in the sustained signaling activity of this chimeric receptor. Ubiquitination patterns, rate of endosomal sorting, and signaling kinetics of EGFR fused with the catalytically inactive mutant of AMSH were reversed to normal. Altogether, the data are consistent with the model whereby short K63-linked polyubiquitin chains but not multimonoubiquitin provide an increased avidity for EGFR interactions with ubiquitin adaptors, thus allowing rapid sorting of activated EGFR to the lysosomal degradation pathway.

The Role of EGF Receptor Ubiquitination in Regulating Its Intracellular Traffic

Progression of activated EGF receptor (EGFR) through the endocytic pathway regulates EGFR signaling. Here we show that a non‐ubiquitinated EGFR mutant, unable to bind the endosomal‐sorting complex required for transport (ESCRT) component, Hrs, is not efficiently targeted onto intraluminal vesicles (ILVs) of multivesicular endosomes/bodies (MVBs). Moreover, ubiquitination and ESCRT engagement of activated EGFR are required for EGF‐stimulated ILV formation. Non‐ubiquitinated EGFRs enter clathrin‐coated tubules emanating from MVBs and show enhanced recycling to the plasma membrane, compared to wild‐type EGFR.

Tyrosine Phosphorylation of the β2 Subunit of Clathrin Adaptor Complex AP-2 Reveals the Role of a Di-leucine Motif in the Epidermal Growth Factor Receptor Trafficking

Tyrosine phosphorylation of the β2 subunit of clathrin adaptor complex AP-2 was detected in three types of cells treated with epidermal growth factor (EGF). The tyrosine phosphorylation was observed during recruitment of EGF receptors into coated pits at 4 °C and reached maximum at 37 °C at post-recruitment stages of endocytosis. An inhibitor of EGF receptor kinase completely abolished this phosphorylation in all cell types, whereas the inhibitor of Src family kinases partially inhibited β2 phosphorylation in A-431 cells but not in HeLa cells. By using β2 subunit tagged with yellow fluorescent protein that is effectively assembled into AP-2 complex, the major phosphorylation site of β2 was mapped to Tyr-6. Analysis of cells expressing dominant-interfering mutant μ2 subunit of AP-2 suggested that β2 phosphorylation is partially mediated by the receptor interaction with the μ2 subunit. Mutation of leucine residues 1010 and 1011 motif in the EGF receptor resulted in the severe inhibition of β2 tyrosine phosphorylation. From these data, we propose that interactions of the EGF receptor with AP-2 mediated by the receptor 974YRAL and di-leucine motifs may contribute to β2 tyrosine phosphorylation. Surprisingly, mutation of the Leu-1010/Leu-1011 motif resulted in impaired degradation of EGF receptors, suggesting the role of this motif in lysosomal targeting of the receptor.