Ligand-induced lysosomal epidermal growth factor receptor (EGFR) degradation is preceded by proteasome-dependent EGFR de-ubiquitination

Studies on the differential routing of internalized epidermal growth factor receptors (EGFRs) induced by EGF, TGF alpha, and the superagonist EGF-TGF alpha chimera E4T suggested a correlation between receptor recycling and their mitogenic potency. EGFR sorting to lysosomes depends on its kinase domain and its ubiquitination by Cbl proteins. Proteasomes have also been proposed to regulate EGFR degradation, but the underlying mechanism remains obscure. Here we evaluated EGFR activation, Cbl recruitment, EGFR ubiquitination and degradation in response to EGF, TGF alpha, and E4T. We also determined the fate of activated EGFRs and Cbl proteins by using v-ATPase (bafilomycin A1) and proteasome (lactacystin) inhibitors. Our results demonstrate that E4T and TGF alpha provoke decreased Cbl recruitment, EGFR ubiquitination and EGFR degradation compared with EGF. Furthermore, bafilomycin treatment blocks EGFR but not c-Cbl degradation. In contrast, lactacystin treatment blocks EGF-induced c-Cbl degradation but does not block EGFR degradation, even though lactacystin causes a minor delay in EGFR degradation. Surprisingly, even though bafilomycin completely blocks EGFR degradation, it does not prevent EGFR de-ubiquitination upon prolonged EGF stimulation. Strikingly, when combined with bafilomycin, lactacystin treatment stabilizes the ubiquitinated EGFR and prevents its de-ubiquitination. We conclude that the enhanced EGFR recycling that has been observed in HER-14 cells following TGF alpha or E4T stimulation correlates with decreased EGFR ubiquitination and EGFR degradation, and that proteasomal activity is required for de-ubiquitination of the EGFR prior to its lysosomal degradation.     

Regulation of epidermal growth factor receptor down-regulation by UBPY-mediated deubiquitination at endosomes

Ligand-activated receptor tyrosine kinases undergo endocytosis and are transported via endosomes to lysosomes for degradation. This "receptor down-regulation" process is crucial to terminate the cell proliferation signals produced by activated receptors. During the process, ubiquitination of the receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. Here, we describe the role of a deubiquitinating enzyme UBPY/USP8 in the down-regulation of epidermal growth factor (EGF) receptor (EGFR). Overexpression of UBPY reduced the ubiquitination level of EGFR and delayed its degradation in EGF-stimulated cells. Immunopurified UBPY deubiquitinated EGFR in vitro. In EGF-stimulated cells, UBPY underwent ubiquitination and bound to EGFR. Overexpression of Hrs or a dominant-negative mutant of SKD1, proteins that play roles in the endosomal sorting of ubiquitinated receptors, caused the accumulation of endogenous UBPY on exaggerated endosomes. A catalytically inactive UBPY mutant clearly localized on endosomes, where it overlapped with EGFR when cells were stimulated with EGF. Finally, depletion of endogenous UBPY by RNA interference resulted in elevated ubiquitination and accelerated degradation of EGF-activated EGFR. We conclude that UBPY negatively regulates the rate of EGFR down-regulation by deubiquitinating EGFR on endosomes.     

Degradation of endocytosed epidermal growth factor and virally ubiquitinated major histocompatibility complex class I is independent of mammalian ESCRTII

Models for protein sorting at multivesicular bodies in the endocytic pathway of mammalian cells have relied largely on data obtained from yeast. These data suggest the essential role of four ESCRT complexes in multivesicular body protein sorting. However, the putative mammalian ESCRTII complex (hVps25p, hVps22p, and hVps36p) has no proven functional role in endosomal transport. We have characterized the human ESCRTII complex and investigated its function in endosomal trafficking. The human ESCRTII proteins interact with one another, with hVps20p (a component of ESCRTIII), and with their yeast homologues. Our interaction data from yeast two-hybrid studies along with experiments with purified proteins suggest an essential role for the N-terminal domain of hVps22p in the formation of a heterotetrameric ESCRTII complex. Although human ESCRTII is found in the cytoplasm and in the nucleus, it can be recruited to endosomes upon overexpression of dominant-negative hVps4Bp. Interestingly, we find that small interference RNA depletion of mammalian ESCRTII does not affect degradation of epidermal growth factor, a known cargo of the multivesicular body protein sorting pathway. We also show that depletion of the deubiquitinating enzymes AMSH (associated molecule with the SH3 domain of STAM (signal transducing adaptor molecule)) and UBPY (ubiquitin isopeptidase Y) have opposite effects on epidermal growth factor degradation, with UBPY depletion causing dramatic swelling of endosomes. Down-regulation of another cargo, the major histocompatibility complex class I in cells expressing the Kaposi sarcoma-associated herpesvirus protein K3, is unaffected in ESCRTII-depleted cells. Our data suggest that mammalian ESCRTII may be redundant, cargo-specific, or not required for protein sorting at the multivesicular body.     

UBE4B Protein Couples Ubiquitination and Sorting Machineries to Enable Epidermal Growth Factor Receptor (EGFR) Degradation

The signaling of plasma membrane proteins is tuned by internalization and sorting in the endocytic pathway prior to recycling or degradation in lysosomes. Ubiquitin modification allows recognition and association of cargo with endosomally associated protein complexes, enabling sorting of proteins to be degraded from those to be recycled. The mechanism that provides coordination between the cellular machineries that mediate ubiquitination and endosomal sorting is unknown. We report that the ubiquitin ligase UBE4B is recruited to endosomes in response to epidermal growth factor receptor (EGFR) activation by binding to Hrs, a key component of endosomal sorting complex required for transport (ESCRT) 0. We identify the EGFR as a substrate for UBE4B, establish UBE4B as a regulator of EGFR degradation, and describe a mechanism by which UBE4B regulates endosomal sorting, affecting cellular levels of the EGFR and its downstream signaling. We propose a model in which the coordinated action of UBE4B, ESCRT-0, and the deubiquitinating enzyme USP8 enable the endosomal sorting and lysosomal degradation of the EGFR.     

Endocytosis: the DUB version

Dynamic modification of endosomal cargo proteins, such as the epidermal growth factor receptor, by ubiquitin can regulate their sorting into the lumen of multivesicular bodies through interactions with a complex protein network incorporating the endosomal sorting complexes required for transport (ESCRTs). Two deubiquitinating enzymes, AMSH and UBPY, interact with ESCRT protein components but exert opposite effects upon the rate of epidermal growth factor receptor downregulation. This might reflect their distinct specificities for different types of polyubiquitin chain linkage. We propose that AMSH might rescue ubiquitinated cargo from lysosomal degradation through disassembly of K63-linked polyubiquitin chains. UBPY function is essential for effective downregulation but is likely to be multifaceted, encompassing activity against both K63-linked and K48-linked polyubiquitin chains and including regulation of the stability of ESCRT-associated proteins such as STAM, by reversing their ubiquitination.     

A deubiquitinating enzyme UBPY regulates the level of protein ubiquitination on endosomes

Monoubiquitination of endocytosed cell surface receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. The sorting of ubiquitinated proteins is executed by concerted actions of class E vacuolar protein sorting (Vps) proteins. Some proteins in the sorting machinery undergo monoubiquitination, suggesting that their functions are also regulated by ubiquitination. The Hrs-STAM complex, a class E Vps protein complex essential for the initial step of the sorting pathway, binds two deubiquitinating enzymes, UBPY and AMSH. Here we examined the effects of inactivating UBPY on protein ubiquitination at endosomes. Overexpression of a catalytically inactive UBPY mutant or depletion of UBPY by RNA interference resulted in the accumulation of ubiquitinated proteins on morphologically aberrant endosomes. Electron microscopy showed that they are aggregates of multivesicular endosomes. Among the sorting machinery proteins that undergo ubiquitination, Eps15 was monoubiquitinated at an elevated level in UBPY-inactivated cells. UBPY also deubiquitinated Eps15 in vitro, suggesting that Eps15 is a cellular substrate for UBPY. Furthermore, inactivation of UBPY caused the accumulation of Eps15 on the endosomal aggregates. These results suggest that UBPY regulates the level of protein ubiquitination on endosomes, which is required for maintaining the morphology of the organelle.     

HER2-mediated effects on EGFR endosomal sorting: analysis of biophysical mechanisms

Overexpression of HER2, a receptor-like tyrosine kinase and signaling partner for the epidermal growth factor receptor (EGFR), has been implicated in numerous experimental and clinical studies as promoting the progression of many types of cancer. One avenue by which HER2 overexpression may dysregulate EGFR-mediated cell responses, such as proliferation and migration, downstream of EGF family ligand binding, is by its modulation on EGFR endocytic trafficking dynamics. EGFR signaling is regulated by downregulation and compartmental relocalization arising from endocytic internalization and endosomal sorting to degradation versus recycling fates. HER2 overexpression influences both of these processes. At the endosomal sorting stage, increased HER2 levels elicit enhanced EGFR recycling outcomes, but the mechanism by which this transpires is poorly understood. Here, we determine whether alternative mechanisms for HER2-mediated enhancement of EGFR recycling can be distinguished by comparison of corresponding mathematical models to experimental literature data. Indeed, we find that the experimental data are clearly most consistent with a mechanism in which HER2 directly competes with EGFR for a stoichiometrically-limited quantity of endosomal retention components (ERCs), thereby reducing degradation of ERC-coupled EGFR. Model predictions based on this mechanism exhibited qualitative trends highly similar to data on the fraction of EGF/EGFR complexes sorted to recycling fate as a function of the amount of internalized EGF/EGFR complexes. In contrast, model predictions for alternative mechanisms-blocking of EGFR/ERC coupling, or altering EGF/EGFR dissociation-were inconsistent with the qualitative trends of the experimental data.     

Insertion-trigger residues differentially modulate endosomal escape by cytotoxic necrotizing factor toxins

The cellular specificity, potency, and modular nature of bacterial protein toxins enable their application for targeted cytosolic delivery of therapeutic cargo. Efficient endosomal escape is a critical step in the design of bacterial toxin-inspired drug delivery (BTIDD) vehicles to avoid lysosomal degradation and promote optimal cargo delivery. The cytotoxic necrotizing factor (CNF) family of modular toxins represents a useful model for investigating cargo-delivery mechanisms due to the availability of many homologs with high sequence identity, their flexibility in swapping domains, and their differential activity profiles. Previously, we found that CNFy is more sensitive to endosomal acidification inhibitors than CNF1 and CNF2. Here, we report that CNF3 is even less sensitive than CNF1/2. We identified two amino acid residues within the putative translocation domain (E374 and E412 in CNFy, Q373 and S411 in CNF3) that differentiate between these two toxins. Swapping these corresponding residues in each toxin changed the sensitivity to endosomal acidification and efficiency of cargo-delivery to be more similar to the other toxin. Results suggested that trafficking to the more acidic late endosome is required for cargo delivery by CNFy but not CNF3. This model was supported by results from toxin treatment of cells in the presence of NH₄Cl, which blocks endosomal acidification, and of small-molecule inhibitors EGA, which blocks trafficking to late endosomes, and ABMA, which blocks endosomal escape and trafficking to the lysosomal degradative pathway. These findings suggest that it is possible to fine-tune endosomal escape and cytosolic cargo delivery efficiency in designing BTIDD platforms.     

Endosomal Deubiquitinating Enzymes Control Ubiquitination and Down-regulation of Protease-activated Receptor 2

The E3 ubiquitin ligase c-Cbl ubiquitinates the G protein-coupled receptor protease-activated receptor 2 (PAR₂), which is required for postendocytic sorting of activated receptors to lysosomes, where degradation terminates signaling. The mechanisms of PAR₂ deubiquitination and its importance in trafficking and signaling of endocytosed PAR₂ are unknown. We report that receptor deubiquitination occurs between early endosomes and lysosomes and involves the endosomal deubiquitinating proteases AMSH and UBPY. Expression of the catalytically inactive mutants, AMSH(D348A) and UBPY(C786S), caused an increase in PAR₂ ubiquitination and trapped the receptor in early endosomes, thereby preventing lysosomal trafficking and degradation. Small interfering RNA knockdown of AMSH or UBPY also impaired deubiquitination, lysosomal trafficking, and degradation of PAR₂. Trapping PAR₂ in endosomes through expression of AMSH(D348A) or UBPY(C786S) did not prolong the association of PAR₂ with β-arrestin2 or the duration of PAR₂-induced ERK2 activation. Thus, AMSH and UBPY are essential for trafficking and down-regulation of PAR₂ but not for regulating PAR₂ dissociation from β-arrestin2 or PAR₂-mediated ERK2 activation.Ubiquitination of certain G protein-coupled receptors (GPCRs)³ is an essential signal for their postendocytic trafficking to lysosomes, which prevents uncontrolled signaling during chronic stimulation. Agonists stimulate ubiquitination of the β₂-adrenergic receptor (β₂AR), chemokine (CXC motif) receptor 4, and protease-activated receptor 2 (PAR₂), and the E3 ubiquitin ligases that mediate ubiquitination of these GPCRs and associated proteins, such as β-arrestins, have been identified (1–3). Although ubiquitination of these receptors is not required for endocytosis, ubiquitin-resistant mutant receptors show diminished postendocytic sorting to lysosomes and impaired down-regulation. However, despite of the reversible nature of this post-translational modification, little is known about the role of deubiquitinating proteases (DUBs) in the postendocytic trafficking and signaling of GPCRs.Our understanding of the role of DUBs in postendocytic receptor trafficking mostly derives from studies of receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR). Two endosomal DUBs, AMSH (associated molecule with the Src homology 3 domain of STAM (signal-transducing adapter molecule)) and UBPY (ubiquitin-specific protease Y) (also known as USP8), regulate deubiquitination and postendocytic trafficking of EGFR (4). AMSH belongs to the JAMM (JAB1/MPN/Mov34) family of metalloproteases and shows specificity for Lys⁶³- over Lys⁴⁸-linked ubiquitin chains (5, 6). UBPY is a cysteine protease of the ubiquitin-specific protease (USP) family and does not discriminate between Lys⁴⁸- and Lys⁶³-linked ubiquitin (7, 8). Activated EGFR recruits the E3 ligase c-Cbl, which ubiquitinates the receptor (9). Ubiquitinated EGFR then interacts with the Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate)-STAM complex in early endosomes (10). Hrs-STAM forms part of the ESCRT (endosomal sorting complex required for transport)-I, -II, -III complex that sorts ubiquitinated receptors in multivesicular bodies (MVBs) to intralumenal vesicles that eventually fuse with lysosomes, where degradation occurs (11). Before receptors are incorporated into the intralumenal vesicles, they are deubiquitinated, which serves to maintain levels of free ubiquitin (11). AMSH and UBPY interact directly with STAM through a common binding site within its Src homology 3 domain (12–14). The balance of EGFR ubiquitination by c-Cbl and deubiquitination by AMSH and UBPY controls the postendocytic trafficking and down-regulation of the EGFR. c-Cbl promotes lysosomal degradation of the EGFR (9), AMSH opposes c-Cbl action and promotes EGFR recycling (5), and UBPY is required for lysosomal sorting and degradation of EGFR (8, 15–17). The role of AMSH and UBPY in regulating deubiquitination, trafficking, and signaling of GPCRs in endosomes is largely unknown. A recent study has shown, however, that AMSH and UBPY regulate the down-regulation of the δ-opioid receptor (DOR), a GPCR that is ubiquitinated and degraded following activation (18). Expression of catalytically inactive mutants of AMSH or UBPY or knockdown of AMSH or UBPY levels using siRNA inhibits down-regulation of DOR. Interestingly, the roles of AMSH and UBPY in DOR down-regulation appear to be nonredundant, since depletion of either DUB produced comparable effects, and simultaneous depletion of both DUBs did not have additional consequences (18). Different DUBs, USP20 and -33, have been recently shown to reverse agonist-induced ubiquitination of the β₂AR (19).We examined the roles of AMSH and UBPY in the ubiquitination, postendocytic trafficking, and lysosomal degradation of PAR₂. We also determined whether AMSH and UBPY regulate PAR₂ association with β-arrestins in endosomes and control β-arrestin-mediated extracellular signal-regulated kinase (ERK) activation. PAR₂ is a receptor for multiple serine proteases that are generated during injury and inflammation (20). Activated PAR₂ promotes inflammation and pain, and PAR₂ contributes to inflammatory diseases of the airway, joints, and intestine. PAR₂ levels are elevated during inflammation, due to increased mRNA expression or perhaps decreased receptor degradation, which amplifies the proinflammatory actions of proteases (21). Given the irreversible nature of proteolytic activation, and since the internalized receptor probably signals by the β-arrestin-dependent recruitment of mitogen-activated protein kinase (MAPK) to endosomes (22), termination of PAR₂ signaling requires receptor degradation in lysosomes, which in turn is ubiquitination-dependent (3, 23). It is therefore important to understand mechanisms of PAR₂ ubiquitination and lysosomal targeting and also how these processes can be reversed. We have reported that activated PAR₂ is monoubiquitinated at multiple sites by the E3 ligase c-Cbl and targeted to lysosomes by an Hrs-dependent pathway (3, 24). Nothing is known about the mechanism and function of PAR₂ deubiquitination. Herein, we examined the role of AMSH and UBPY in regulating the deubiquitination, lysosomal trafficking, and degradation of PAR₂, the interaction of PAR₂ with β-arrestin2, and β-arrestin-mediated ERK2 activation. We demonstrate that endosomal DUBs are key regulators of GPCR down-regulation.     

Aberrant trafficking of NSCLC-associated EGFR mutants through the endocytic recycling pathway promotes interaction with Src@

Background  

Epidermal growth factor receptor (EGFR) controls a wide range of cellular processes, and altered EGFR signaling contributes to human cancer. EGFR kinase domain mutants found in non-small cell lung cancer (NSCLC) are constitutively active, a trait critical for cell transformation through activation of downstream pathways. Endocytic trafficking of EGFR is a major regulatory mechanism as ligand-induced lysosomal degradation results in termination of signaling. While numerous studies have examined mutant EGFR signaling, the endocytic traffic of mutant EGFR within the NSCLC milieu remains less clear.     

Epidermal growth factor receptor fate is controlled by Hrs tyrosine phosphorylation sites that regulate Hrs degradation

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is an endosomal protein essential for the efficient sorting of activated growth factor receptors into the lysosomal degradation pathway. Hrs undergoes ligand-induced tyrosine phosphorylation on residues Y329 and Y334 downstream of epidermal growth factor receptor (EGFR) activation. It has been difficult to investigate the functional roles of phosphoHrs, as only a small proportion of the cellular Hrs pool is detectably phosphorylated. Using an HEK 293 model system, we found that ectopic expression of the protein Cbl enhances Hrs ubiquitination and increases Hrs phosphorylation following cell stimulation with EGF. We exploited Cbl's expansion of the phosphoHrs pool to determine whether Hrs tyrosine phosphorylation controls EGFR fate. In structure-function studies of Cbl and EGFR mutants, the level of Hrs phosphorylation and rapidity of apparent Hrs dephosphorylation correlated directly with EGFR degradation. Differential expression of wild-type versus Y329,334F mutant Hrs in Hrs-depleted cells revealed that one or both tyrosines regulate ligand-dependent Hrs degradation, as well as EGFR degradation. By modulating Hrs ubiquitination, phosphorylation, and protein levels, Cbl may control the composition of the endosomal sorting machinery and its ability to target EGFR for lysosomal degradation.     

A role for ADP ribosylation factor in the control of cargo uptake during COPI-coated vesicle biogenesis

ARF-mediated hydrolysis of GTP has been demonstrated to regulate coat disassembly of Golgi-derived COPI transport vesicles (Tanigawa, G., Orci, L., Amherdt, M., Ravazzola, M., Helms, J.B. and Rothman, J.E. (1993) J. Cell Biol. 123, 1365-1371). In addition, a requirement for GTP hydrolysis at an early stage of COPI vesicle biogenesis has been established since cargo uptake is impaired in the presence of GTPgammaS (Nickel, W., Malsam, J., Gorgas, K., Ravazzola, M., Jenne, N., Helms, J.B. and Wieland, F.T. (1998) J. Cell Sci. 111, 3081-3090), a non-hydrolyzable analogue of GTP. We now demonstrate that the GTPase involved in the regulation of cargo uptake is ARF, revealing a multi-functional role of this GTPase in COPI-mediated vesicular transport. The molecular mechanism of cargo uptake as well as the functional implications of these findings on the overall process of COPI vesicle biogenesis are discussed.     

EGFR-dependent phosphorylation of leucine-rich repeat kinase LRRK1 is important for proper endosomal trafficking of EGFR

Ligand-induced activation of the epidermal growth factor receptor (EGFR) initiates trafficking events that relocalize the receptors from the cell surface to intracellular endocytic compartments. We recently reported that leucine-rich repeat kinase 1 (LRRK1) is involved in the trafficking of EGFR from early to late endosomes. In this study, we demonstrate that EGFR regulates the kinase activity of LRRK1 via tyrosine phosphorylation and that this is required for proper endosomal trafficking of EGFR. Phosphorylation of LRRK1 at Tyr-944 results in reduced LRRK1 kinase activity. Mutation of LRRK1 Tyr-944 (Y944F) abolishes EGF-stimulated tyrosine phosphorylation, resulting in hyperactivation of LRRK1 kinase activity and enhanced motility of EGF-containing endosomes toward the perinuclear region. The compartments in which EGFR accumulates are mixed endosomes and are defective in the proper formation of intraluminal vesicles of multivesicular bodies. These results suggest that feedback down-regulation of LRRK1 kinase activity by EGFR plays an important role in the appropriate endosomal trafficking of EGFR.     

LAPTM4B is a PtdIns(4,5)P2 effector that regulates EGFR signaling,lysosomal sorting,and degradation

Lysosomal degradation is essential for the termination of EGF‐stimulated EGF receptor (EGFR) signaling. This requires EGFR sorting to the intraluminal vesicles (ILVs) of multi‐vesicular endosomes (MVEs). Cytosolic proteins including the ESCRT machineries are key regulators of EGFR intraluminal sorting, but roles for endosomal transmembrane proteins in receptor sorting are poorly defined. Here, we show that LAPTM4B, an endosomal transmembrane oncoprotein, inhibits EGF‐induced EGFR intraluminal sorting and lysosomal degradation, leading to enhanced and prolonged EGFR signaling. LAPTM4B blocks EGFR sorting by promoting ubiquitination of Hrs (an ESCRT‐0 subunit), which inhibits the Hrs association with ubiquitinated EGFR. This is counteracted by the endosomal PIP kinase, PIPKIγi5, which directly binds LAPTM4B and neutralizes the inhibitory function of LAPTM4B in EGFR sorting by generating PtdIns(4,5)P₂ and recruiting SNX5. PtdIns(4,5)P₂ and SNX5 function together to protect Hrs from ubiquitination, thereby promoting EGFR intraluminal sorting. These results reveal an essential layer of EGFR trafficking regulated by LAPTM4B, PtdIns(4,5)P₂ signaling, and the ESCRT complex and define a mechanism by which the oncoprotein LAPTM4B can transform cells and promote tumor progression.     

Biogenesis of Lysosome-related Organelles Complex-1 Subunit 1 (BLOS1) Interacts with Sorting Nexin 2 and the Endosomal Sorting Complex Required for Transport-I (ESCRT-I) Component TSG101 to Mediate the Sorting of Epidermal Growth Factor Receptor into Endosomal Compartments

Biogenesis of lysosome-related organelles complex-1 (BLOC-1) is a component of the molecular machinery required for the biogenesis of specialized organelles and lysosomal targeting of cargoes via the endosomal to lysosomal trafficking pathway. BLOS1, one subunit of BLOC-1, is implicated in lysosomal trafficking of membrane proteins. We found that the degradation and trafficking of epidermal growth factor receptor (EGFR) were delayed in BLOS1 knockdown cells, which were rescued through BLOS1 overexpression. A key feature to the delayed EGFR degradation is the accumulation of endolysosomes in BLOS1 knockdown cells or BLOS1 knock-out mouse embryonic fibroblasts. BLOS1 interacted with SNX2 (a retromer subunit) and TSG101 (an endosomal sorting complex required for transport subunit-I) to mediate EGFR lysosomal trafficking. These results suggest that coordination of the endolysosomal trafficking proteins is important for proper targeting of EGFR to lysosomes.     

Characterization of the insulin-regulated endocytic recycling mechanism in 3T3-L1 adipocytes using a novel reporter molecule

The endocytic trafficking of the GLUT4 glucose transporter and the insulin-regulated aminopeptidase (IRAP) are regulated by insulin. We have used a chimera between the intracellular domain of IRAP and the extracellular and transmembrane domains of the transferrin receptor (vpTR) to characterize IRAP-like trafficking in 3T3-L1 adipocytes. Our data demonstrate that the cytoplasmic domain of IRAP is sufficient to target vpTR to the insulin-regulated, slow recycling pathway in adipocytes and that the dynamic retention of vpTR is dependent on a di-leucine motif. Our kinetic analysis demonstrates that vpTR recycles as a single kinetic pool and that vpTR is very efficiently sorted from endosomes to the insulin-regulated recycling pathway. An implication of these findings is that the key step in the dynamic retention of vpTR occurs within the early endosomal system. We have previously shown that vpTR is trafficked by an insulin-regulated pathway in Chinese hamster ovary cells (Johnson, A. O., Subtil, A., Petrush, R., Kobylarz, K., Keller, S., and Mc Graw, T. E. (1998) J. Biol. Chem. 273, 17968-17977). The behavior of vpTR in Chinese hamster ovary cells is similar to its behavior in 3T3-L1 adipocytes. The main difference is that insulin has a larger effect on the trafficking of vpTR in the adipocytes. We concluded that the insulin-regulated slow recycling endocytic mechanism is expressed in many different cell types and therefore is not a unique characteristic of cells that express GLUT4.     

Presenilin 1 regulates epidermal growth factor receptor turnover and signaling in the endosomal-lysosomal pathway

Mutations in the gene encoding presenilin 1 (PS1) cause the most aggressive form of early-onset familial Alzheimer disease. In addition to its well established role in Abeta production and Notch proteolysis, PS1 has been shown to mediate other physiological activities, such as regulation of the Wnt/beta-catenin signaling pathway, modulation of phosphatidylinositol 3-kinase/Akt and MEK/ERK signaling, and trafficking of select membrane proteins and/or intracellular vesicles. In this study, we present evidence that PS1 is a critical regulator of a key signaling receptor tyrosine kinase, epidermal growth factor receptor (EGFR). Specifically, EGFR levels were robustly increased in fibroblasts deficient in both PS1 and PS2 (PS(-/-)) due to delayed turnover of EGFR protein. Stable transfection of wild-type PS1 but not PS2 corrected EGFR to levels comparable to PS(+/+) cells, while FAD PS1 mutations showed partial loss of activity. The C-terminal fragment of PS1 was sufficient to fully reduce EGFR levels. In addition, the rapid ligand-induced degradation of EGFR was markedly delayed in PS(-/-) cells, resulting in prolonged signal activation. Despite the defective turnover of EGFR, ligand-induced autophosphorylation, ubiquitination, and endocytosis of EGFR were not affected by the lack of PS1. Instead, the trafficking of EGFR from early endosomes to lysosomes was severely delayed by PS1 deficiency. Elevation of EGFR was also seen in brains of adult mice conditionally ablated in PS1 and in skin tumors associated with the loss of PS1. These findings demonstrate a critical role of PS1 in the trafficking and turnover of EGFR and suggest potential pathogenic effects of elevated EGFR as well as perturbed endosomal-lysosomal trafficking in cell cycle control and Alzheimer disease.     

The PtdIns3P‐Binding Protein Phafin 2 Mediates Epidermal Growth Factor Receptor Degradation by Promoting Endosome Fusion

Phosphatidylinositol 3‐phosphate (PtdIns3P) orchestrates endosomal cargo transport, fusion and motility by recruiting FYVE or PX domain‐containing effector proteins to endosomal membranes. In an attempt to discover novel PtdIns3P effectors involved in the termination of growth factor receptor signalling, we performed an siRNA screen for epidermal growth factor (EGF) degradation, targeting FYVE and PX domain proteins in the human proteome. This screen identified several potential regulators of EGF degradation, including HRS (used as positive control), PX kinase, MTMR4 and Phafin2/PLEKHF2. As Phafin2 has not previously been shown to be required for EGF receptor (EGFR) degradation, we performed further functional studies on this protein. Loss of Phafin2 was found to decrease early endosome size, whereas overexpression of Phafin2 resulted in enlarged endosomes. Moreover, both the EGFR and the fluid‐phase marker dextran were retained in abnormally small endosomes in Phafin2‐depleted cells. In yeast two‐hybrid analysis we identified Phafin2 as a novel interactor of the endosomal‐tethering protein EEA1, and Phafin2 colocalized strongly with EEA1 in microdomains of the endosome membrane. Our results suggest that Phafin2 controls receptor trafficking and fluid‐phase transport through early endosomes by facilitating endosome fusion in concert with EEA1.