Alexander Sorkin

Endocytosis and signalling: intertwining molecular networks

Cell signalling and endocytic membrane trafficking have traditionally been viewed as distinct processes. Although our present understanding is incomplete and there are still great controversies, it is now recognized that these processes are intimately and bidirectionally linked in animal cells. Indeed, many recent examples illustrate how endocytosis regulates receptor signalling (including signalling from receptor tyrosine kinases and G protein-coupled receptors) and, conversely, how signalling regulates the endocytic pathway. The mechanistic and functional principles that underlie the relationship between signalling and endocytosis in cell biology are becoming increasingly evident across many systems.

Signal transduction and endocytosis: close encounters of many kinds

Binding of hormones, growth factors and other cell modulators to cell-surface receptors triggers a complex array of signal-transduction events. The activation of many receptors also accelerates their endocytosis. Endocytic transport is important in regulating signal transduction and in mediating the formation of specialized signalling complexes. Conversely, signal-transduction events modulate specific components of the endocytic machinery. Recent studies of protein tyrosine kinases and G-protein-coupled receptors have shed new light on the mechanisms and functional consequences of this bidirectional interplay between signalling and membrane-transport networks.

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.

Eps15 Is a Component of Clathrin-coated Pits and Vesicles and Is Located at the Rim of Coated Pits

Eps15, a phosphorylation substrate of the epidermal growth factor (EGF) receptor kinase, has been shown to bind to the α-subunit of the clathrin-associated protein complex AP-2. Here we report that in cells, virtually all Eps15 interacts with the cytosol and membrane-bound forms of AP-2. This association is not affected by the treatment of cells with EGF. Immunofluorescence microscopy reveals nearly absolute co-localization of Eps15 with AP-2 and clathrin, and analysis by immunoelectron microscopy shows that the localization of membrane-associated Eps15 is restricted to the profiles corresponding to endocytic coated pits and vesicles. Unexpectedly, Eps15 was found at the edge of forming coated pits and at the rim of budding coated vesicles. This asymmetric distribution is in sharp contrast to the localization of AP-2 that shows an even distribution along the same types of clathrin-coated structures. These findings suggest several possible regulatory roles of Eps15 during the formation of coated pits.

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.

Endocytosis of Receptor Tyrosine Kinases

Endocytosis is the major regulator of signaling from receptor tyrosine kinases (RTKs). The canonical model of RTK endocytosis involves rapid internalization of an RTK activated by ligand binding at the cell surface and subsequent sorting of internalized ligand-RTK complexes to lysosomes for degradation. Activation of the intrinsic tyrosine kinase activity of RTKs results in autophosphorylation, which is mechanistically coupled to the recruitment of adaptor proteins and conjugation of ubiquitin to RTKs. Ubiquitination serves to mediate interactions of RTKs with sorting machineries both at the cell surface and on endosomes. The pathways and kinetics of RTK endocytic trafficking, molecular mechanisms underlying sorting processes, and examples of deviations from the standard trafficking itinerary in the RTK family are discussed in this work.

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.

Three-chromophore FRET microscopy to analyze multiprotein interactions in living cells

Nearly every major process in a cell is carried out by assemblies of multiple dynamically interacting protein molecules. To study multi-protein interactions within such molecular machineries, we have developed a fluorescence microscopy method called three-chromophore fluorescence resonance energy transfer (3-FRET). This method allows analysis of three mutually dependent energy transfer processes between the fluorescent labels, such as cyan, yellow and monomeric red fluorescent proteins. Here, we describe both theoretical and experimental approaches that discriminate the parallel versus the sequential energy transfer processes in the 3-FRET system. These approaches were established in vitro and in cultured mammalian cells, using chimeric proteins consisting of two or three fluorescent proteins linked together. The 3-FRET microscopy was further applied to the analysis of three-protein interactions in the constitutive and activation-dependent complexes in single endosomal compartments. These data highlight the potential of 3-FRET microscopy in studies of spatial and temporal regulation of signaling processes in living cells.

RNA Interference Screen Reveals an Essential Role of Nedd4–2 in Dopamine Transporter Ubiquitination and Endocytosis

The function of the dopamine transporter (DAT) to terminate dopamine neurotransmission is regulated by endocytic trafficking of DAT. To elucidate the mechanisms of DAT endocytosis, we generated a fully functional mutant of the human DAT in which a hemagglutinin epitope (HA) was incorporated into the second extracellular loop. The endocytosis assay, based on the uptake of an HA antibody, was designed to study constitutive- and protein kinase C (PKC)-dependent internalization of HA-DAT expressed in non-neuronal cells and rat dopaminergic neurons. Large-scale RNA interference analysis of PKC-dependent endocytosis of HA-DAT revealed the essential and specific role of an E3 ubiquitin ligase, Nedd4–2 (neural precursor cell expressed, developmentally downregulated 4–2), as well as the involvement of adaptor proteins present in clathrin-coated pits, such as epsin, Eps15 (epidermal growth factor pathway substrate clone 15), and Eps15R (Eps15-related protein). Depletion of Nedd4–2 resulted in a dramatic reduction of PKC-dependent ubiquitination of DAT. Endogenous Nedd4–2, epsin, and Eps15 were coimmunoprecipitated with heterologously expressed human HA-DAT and endogenous DAT isolated from rat striatum. A new mechanistic model of DAT endocytosis is proposed whereby the PKC-induced ubiquitination of DAT mediated by Nedd4–2 leads to interaction of DAT with adaptor proteins in coated pits and acceleration of DAT endocytosis.

Epidermal Growth Factor Receptor Interaction with Clathrin Adaptors Is Mediated by the Tyr974-containing Internalization Motif

The carboxyl-terminal regulatory domain of the epidermal growth factor (EGF) receptor is essential for its endocytosis and interaction with the clathrin-associated protein complex AP-2. To identify AP-2 binding motif in the receptor, several single and multiple-point mutations within the region between residues 966 and 977 of the human EGF receptor were made, and the mutant receptors were expressed in NIH3T3 cells. Mutation of tyrosine 974 alone or together with surrounding residues and the deletion of residues 973-975 essentially eliminated AP-2 co-immunoprecipitation with the EGF receptor. Furthermore, a synthetic peptide corresponding to receptor residues 964-978 blocked AP-2 association with the wild-type EGF receptor. These data suggest that AP-2 has only one high-affinity binding site in the EGF receptor composed of Tyr974-containing motif. Receptor mutants that did not bind AP-2 displayed a lower rate of internalization, down-regulation, and turnover compared to wild-type receptors when expressed at high levels. However, similar receptor mutants expressed at low levels were internalized and down-regulated as efficiently as wild-type receptors. Internalization of the mutant receptors lacking the high-affinity binding site for AP-2 was inhibited by K+-depletion of the cells, indicating that their endocytosis required intact coated pits. We suggest that whereas one mechanism of EGF receptor recruitment into coated pits involves high-affinity binding of AP-2 to Tyr974-containing motif, another pathway may be mediated by weak receptor/AP-2 interactions or by proteins other than AP-2.