Phosphatidylinositol 4,5-bisphosphate and Arf6-regulated membrane traffic

ADP-ribosylation factor (Arf) 6 regulates the movement of membrane between the plasma membrane (PM) and a nonclathrin-derived endosomal compartment and activates phosphatidylinositol 4-phosphate 5-kinase (PIP 5-kinase), an enzyme that generates phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we show that PIP2 visualized by expressing a fusion protein of the pleckstrin homology domain from PLCdelta and green fluorescent protein (PH-GFP), colocalized with Arf6 at the PM and on tubular endosomal structures. Activation of Arf6 by expression of its exchange factor EFA6 stimulated protrusion formation, the uptake of PM into macropinosomes enriched in PIP2, and recycling of this membrane back to the PM. By contrast, expression of Arf6 Q67L, a GTP hydrolysis-resistant mutant, induced the formation of PIP2-positive actin-coated vacuoles that were unable to recycle membrane back to the PM. PM proteins, such as beta1-integrin, plakoglobin, and major histocompatibility complex class I, that normally traffic through the Arf6 endosomal compartment became trapped in this vacuolar compartment. Overexpression of human PIP 5-kinase alpha mimicked the effects seen with Arf6 Q67L. These results demonstrate that PIP 5-kinase activity and PIP2 turnover controlled by activation and inactivation of Arf6 is critical for trafficking through the Arf6 PM-endosomal recycling pathway.     

GGA3 functions as a switch to promote Met receptor recycling, essential for sustained ERK and cell migration

Cells are dependent on correct sorting of activated receptor tyrosine kinases (RTKs) for the outcome of growth factor signaling. Upon activation, RTKs are coupled through the endocytic machinery for degradation or recycled to the cell surface. However, the molecular mechanisms governing RTK recycling are poorly understood. Here, we show that Golgi-localized gamma ear-containing Arf-binding protein 3 (GGA3) interacts selectively with the Met/hepatocyte growth factor RTK when stimulated, to sort it for recycling in association with "gyrating" clathrin. GGA3 loss abrogates Met recycling from a Rab4 endosomal subdomain, resulting in pronounced trafficking of Met toward degradation. Decreased Met recycling attenuates ERK activation and cell migration. Met recycling, sustained ERK activation, and migration require interaction of GGA3 with Arf6 and an unexpected association with the Crk adaptor. The data show that GGA3 defines an active recycling pathway and support a broader role for GGA3-mediated cargo selection in targeting receptors destined for recycling.     

Clathrin-independent endocytosis: a unique platform for cell signaling and PM remodeling

There is increasing interest in endocytosis that occurs independently of clathrin coats and the fates of membrane proteins internalized by this mechanism. The appearance of clathrin-independent endocytic and membrane recycling pathways seems to vary with different cell types and cargo molecules. In this review we focus on studies that have been performed using HeLa and COS cells as model systems for understanding this membrane trafficking system. These endosomal membranes contain signaling molecules including H-Ras, Rac1, Arf6 and Rab proteins, and a lipid environment rich in cholesterol and PIP(2) providing a unique platform for cell signaling. Furthermore, activation of some of these signaling molecules (H-Ras, Rac and Arf6) can switch the constitutive form of clathrin-independent endocytosis into a stimulated one, associated with PM ruffling and macropinocytosis.     

Sorting of Clathrin‐Independent Cargo Proteins Depends on Rab35 Delivered by Clathrin‐Mediated Endocytosis

Clathrin‐mediated endocytosis (CME) and clathrin‐independent endocytosis (CIE) co‐exist in most cells but little is known about their communication and coordination. Here we show that when CME was inhibited, endocytosis by CIE continued but endosomal trafficking of CIE cargo proteins was altered. CIE cargo proteins that normally traffic directly into Arf6‐associated tubules after internalization and avoid degradation (CD44, CD98 and CD147) now trafficked to lysosomes and were degraded. The endosomal tubules were also absent and Arf6‐GTP levels were elevated. The altered trafficking, loss of the tubular endosomal network and elevated Arf6‐GTP levels caused by inhibition of CME were rescued by expression of Rab35, a Rab associated with clathrin‐coated vesicles, or its effector ACAPs, Arf6 GTPase activating proteins (GAP) that inactivate Arf6. Furthermore, siRNA knockdown of Rab35 recreated the phenotype of CME ablation on CIE cargo trafficking without altering endocytosis of transferrin. These observations suggest that Rab35 serves as a CME detector and that loss of CME, or Rab35 input, leads to elevated Arf6‐GTP and shifts the sorting of CIE cargo proteins to lysosomes and degradation.      

EFA6A,an Exchange Factor for Arf6, Regulates NGF-Dependent TrkA Recycling From Early Endosomes and Neurite Outgrowth in PC12 Cells

Endosomal trafficking of TrkA is a critical process for nerve growth factor (NGF)-dependent neuronal cell survival and differentiation. The small GTPase ADP-ribosylation factor 6 (Arf6) is implicated in NGF-dependent processes in PC12 cells through endosomal trafficking and actin cytoskeleton reorganization. However, the regulatory mechanism for Arf6 in NGF signaling is largely unknown. In this study, we demonstrated that EFA6A, an Arf6-specific guanine nucleotide exchange factor, was abundantly expressed in PC12 cells and that knockdown of EFA6A significantly inhibited NGF-dependent Arf6 activation, TrkA recycling from early endosomes to the cell surface, prolonged ERK1/2 phosphorylation, and neurite outgrowth. We also demonstrated that EFA6A forms a protein complex with TrkA through its N-terminal region, thereby enhancing its catalytic activity for Arf6. Similarly, we demonstrated that EFA6A forms a protein complex with TrkA in cultured dorsal root ganglion (DRG) neurons. Furthermore, cultured DRG neurons from EFA6A knockout mice exhibited disturbed NGF-dependent TrkA trafficking compared with wild-type neurons. These findings provide the first evidence for EFA6A as a key regulator of NGF-dependent TrkA trafficking and signaling.     

A role for EHD4 in the regulation of early endosomal transport

All four of the C-terminal Eps15 homology domain (EHD) proteins have been implicated in the regulation of endocytic trafficking. However, the high level of amino acid sequence identity among these proteins has made it challenging to elucidate the precise function of individual EHD proteins. We demonstrate here with specific peptide antibodies that endogenous EHD4 localizes to Rab5-, early embryonic antigen 1 (EEA1)- and Arf6-containing endosomes and colocalizes with internalized transferrin in the cell periphery. Knock-down of EHD4 expression by both small interfering RNA and short hairpin RNA leads to the generation of enlarged early endosomal structures that contain Rab5 and EEA1 as well as internalized transferrin or major histocompatibility complex class I molecules. In addition, cargo destined for degradation, such as internalized low-density lipoprotein, also accumulates in the enlarged early endosomes in EHD4-depleted cells. Moreover, we have demonstrated that these enlarged early endosomes are enriched in levels of the activated GTP-bound Rab5. Finally, we show that endogenous EHD4 and EHD1 interact in cells, suggesting coordinated involvement in the regulation of receptor transport along the early endosome to endocytic recycling compartment axis. The results presented herein provide evidence that EHD4 is involved in the control of trafficking at the early endosome and regulates exit of cargo toward both the recycling compartment and the late endocytic pathway.     

Syndecan recycling [corrected] is controlled by syntenin-PIP2 interaction and Arf6

Syndecans are heparan sulfate proteoglycans that modulate the activity of several growth factors and cell adhesion molecules. PDZ domains in the adaptor protein syntenin interact with syndecans and with the phosphoinositide PIP(2), which is involved in the regulation of the actin cytoskeleton and membrane trafficking. Here, we show that the syntenin PDZ domain-PIP(2) interaction controls Arf6-mediated syndecan recycling through endosomal compartments. FGF receptor accompanies syndecan along the syntenin-mediated recycling pathway, in a heparan sulfate- and FGF-dependent manner. Syndecans that cannot recycle via this pathway become trapped intracellularly and inhibit cell spreading. This syntenin-mediated syndecan recycling pathway may regulate the surface availability of a number of cell adhesion and signaling molecules.     

Recognising the signals for endosomal trafficking

The endosomal compartment is a major sorting station controlling the balance between endocytic recycling and lysosomal degradation, and its homeostasis is emerging as a central factor in various neurodegenerative diseases such as Alzheimer's and Parkinson's. Membrane trafficking is generally coordinated by the recognition of specific signals in transmembrane protein cargos by different transport machineries. A number of different protein trafficking complexes are essential for sequence-specific recognition and retrieval of endosomal cargos, recycling them to other compartments and acting to counter-balance the default endosomal sorting complex required for transport–mediated degradation pathway. In this review, we provide a summary of the key endosomal transport machineries, and the molecular mechanisms by which different cargo sequences are specifically recognised.     

Pre-sorting endosomal transport of the GPI-anchored protein, CD59, is regulated by EHD1

EHD1 regulates the trafficking of multiple receptors from the endocytic recycling compartment (ERC) to the plasma membrane. However, the potential role of EHD1 in regulating the family of glycosylphosphatidylinositol-anchored proteins (GPI-APs) has not been determined. Here we demonstrate a novel role for EHD1 in regulating the trafficking of CD59, an endogenous GPI-AP, at early stages of trafficking through the endocytic pathway. EHD1 displays significant colocalization with newly internalized CD59. Upon EHD1 depletion, there is a rapid Rab5-independent coalescence of CD59 in the ERC region. However, expression of an active Arf6 mutant (Q67L), which traps internalized pre-sorting endosomal cargo in phosphatidylinositol(4,5)-bisphosphate enriched vacuoles, prevents this coalescence. It is of interest that sustained PKC activation leads to a similar coalescence of CD59 at the ERC, and treatment of EHD1-depleted cells with a PKC inhibitor (Go6976) blocked this rapid relocation of CD59. However, unlike sustained PKC activation, EHD1 depletion does not induce the translocation of PKCα to ERC. The results presented herein provide evidence that EHD1 is involved in the control of CD59 transport from pre-sorting endosomes to the ERC in a PKC-dependent manner. However, the mechanisms of EHD1-induced coalescence of CD59 at the ERC differ from those induced by sustained PKC activation.     

Type II transforming growth factor-beta receptor recycling is dependent upon the clathrin adaptor protein Dab2

Transforming growth factor (TGF)-β family proteins form heteromeric complexes with transmembrane serine/threonine kinases referred to as type I and type II receptors. Ligand binding initiates a signaling cascade that generates a variety of cell type-specific phenotypes. Whereas numerous studies have investigated the regulatory activities controlling TGF-β signaling, there is relatively little information addressing the endocytic and trafficking itinerary of TGF-β receptor subunits. In the current study we have investigated the role of the clathrin-associated sorting protein Disabled-2 (Dab2) in TGF-β receptor endocytosis. Although small interfering RNA-mediated Dab2 knockdown had no affect on the internalization of various clathrin-dependent (i.e., TGF-β, low-density lipoprotein, or transferrin) or -independent (i.e., LacCer) cargo, TGF-β receptor recycling was abrogated. Loss of Dab2 resulted in enlarged early endosomal antigen 1-positive endosomes, reflecting the inability of cargo to traffic from the early endosome to the endosomal recycling compartment and, as documented previously, diminished Smad2 phosphorylation. The results support a model whereby Dab2 acts as a multifunctional adaptor in mesenchymal cells required for TGF-β receptor recycling as well as Smad2 phosphorylation.     

V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway

The recruitment of the small GTPase Arf6 and ARNO from cytosol to endosomal membranes is driven by V-ATPase-dependent intra-endosomal acidification. The molecular mechanism that mediates this pH-sensitive recruitment and its role are unknown. Here, we demonstrate that Arf6 interacts with the c-subunit, and ARNO with the a2-isoform of V-ATPase. The a2-isoform is targeted to early endosomes, interacts with ARNO in an intra-endosomal acidification-dependent manner, and disruption of this interaction results in reversible inhibition of endocytosis. Inhibition of endosomal acidification abrogates protein trafficking between early and late endosomal compartments. These data demonstrate the crucial role of early endosomal acidification and V-ATPase/ARNO/Arf6 interactions in the regulation of the endocytic degradative pathway. They also indicate that V-ATPase could modulate membrane trafficking by recruiting and interacting with ARNO and Arf6; characteristics that are consistent with the role of V-ATPase as an essential component of the endosomal pH-sensing machinery.     

Convergence of non-clathrin- and clathrin-derived endosomes involves Arf6 inactivation and changes in phosphoinositides

The trafficking of two plasma membrane (PM) proteins that lack clathrin internalization sequences, major histocompatibility complex class I (MHCI), and interleukin 2 receptor alpha subunit (Tac) was compared with that of PM proteins internalized via clathrin. MHCI and Tac were internalized into endosomes that were distinct from those containing clathrin cargo. At later times, a fraction of these internalized membranes were observed in Arf6-associated, tubular recycling endosomes whereas another fraction acquired early endosomal autoantigen 1 (EEA1) before fusion with the "classical" early endosomes containing the clathrin-dependent cargo, LDL. After convergence, cargo molecules from both pathways eventually arrived, in a Rab7-dependent manner, at late endosomes and were degraded. Expression of a constitutively active mutant of Arf6, Q67L, caused MHCI and Tac to accumulate in enlarged PIP(2)-enriched vacuoles, devoid of EEA1 and inhibited their fusion with clathrin cargo-containing endosomes and hence blocked degradation. By contrast, trafficking and degradation of clathrin-cargo was not affected. A similar block in transport of MHCI and Tac was reversibly induced by a PI3-kinase inhibitor, implying that inactivation of Arf6 and acquisition of PI3P are required for convergence of endosomes arising from these two pathways.     

Discovery of New Cargo Proteins that Enter Cells through Clathrin-Independent Endocytosis

Clathrin-independent endocytosis (CIE) allows internalization of plasma membrane proteins lacking clathrin-targeting sequences, such as the major histocompatibility complex class I protein (MHCI), into cells. After internalization, vesicles containing MHCI fuse with transferrin-containing endosomes generated from clathrin-dependent endocytosis. In HeLa cells, MHCI is subsequently routed to late endosomes or recycled back out to the plasma membrane (PM) in distinctive tubular carriers. Arf6 is associated with endosomal membranes carrying CIE cargo and expression of an active form of Arf6 leads to the generation of vacuolar structures that trap CIE cargo immediately after endocytosis, blocking the convergence with transferrin-containing endosomes. We isolated these trapped vacuolar structures and analyzed their protein composition by mass spectrometry. Here we identify and validate six new endogenous cargo proteins (CD44, CD55, CD98, CD147, Glut1, and ICAM1) that use CIE to enter cells. CD55 and Glut1 appear to closely parallel the trafficking of MHCI, merging with transferrin endosomes before entering the recycling tubules. In contrast, CD44, CD98, and CD147 appear to directly enter the recycling tubules and by-pass the merge with EEA1-positive, transferrin-containing endosomes. This divergent itinerary suggests that sorting may occur along this CIE pathway. Furthermore, the identification of new cargo proteins will assist others studying CIE in different cell types and tissues.     

Arf6 promotes cell proliferation via the PLD-mTORC1 and p38MAPK pathways

The small G-protein ADP-ribosylation factor 6 (Arf6) belongs to the Ras GTPases superfamily and is mostly known for its actin remodeling functions and involvement in the processes of plasma membrane reorganization and vesicular transport. The majority of data indicates that Arf6 contributes to cancer progression through activation of cell motility and invasion. Alternatively, we found that the expression of a wild-type or a constitutively active Arf6 does not influence tumor cell motility and invasion but instead significantly stimulates cell proliferation and activates phospholipase D (PLD). Conversely the expression of a mutant Arf6 (Arf6N48I), that is, unable to interact with PLD has no effect on proliferation but promotes motility, invasion, and matrix degradation by uPA extracellular proteinase. Studying the mechanisms of Arf6-dependent stimulation of cell proliferation, we found some signaling pathways contributing to Arf6 promitogenic activity. Namely, we showed that Arf6 in a PLD-mTORC1-dependent manner activates S6K1 kinase, a well-known regulator of mitogen-stimulated translation initiation. Furthermore, we demonstrated an Arf6-dependent phosphorylation of mTORC1 downstream targets, 4E-BP1 and ribosomal S6 protein, confirming an existence of Arf6-PLD-mTORC1-S6K1/4E-BP1 signaling pathway and also demonstrated its impact on proliferation stimulation. Next, we found that Arf6 activation potentiates Erk1/2 and p38MAP kinases phosphorylation. Surprisingly, p38 opposite to Erk1/2 significantly contributes to Arf6-dependent proliferation increase promoting S6 ribosomal protein phosphorylation at Ser235/236 residues. Therefore, we demonstrated Arf6 proliferation stimulating activity and revealed PLD-mTORC1 and p38MAP kinase as Arf6 partners mediating promitogenic activity. These results highlight a new aspect of Arf6 functioning in cancer cell biology.     

Crucial roles of Rab22a in endosomal cargo recycling

Endosomal cargo recycling lies at the heart of subcellular trafficking processes under the management of several Ras-related GTP-binding proteins (Rabs) which are coordinated by their upstream regulators and require their downstream effectors to display their functions. In this regard, several Rabs have been well-reviewed except Rab22a. Rab22a is a crucial regulator of vesicle trafficking, early endosome and recycling endosome formation. Notably, recent studies demonstrated the immunological roles of Rab22a, which are closely associated with cancers, infection and autoimmune disorders. This review provides an overview of the regulators and effectors of Rab22a. Also, we highlight the current knowledge of the role of Rab22a in endosomal cargo recycling, including the biogenesis of recycling tubules with the help of a complex with Rab22a at its core, and how different internalized cargo chooses different recycling routes thanks to the cooperation of Rab22a, its effectors and its regulators. Of note, contradictions and speculation related to endosomal cargo recycling that Rab22a brings impacts on are also discussed. Finally, this review endeavors to briefly introduce the various events impacted by Rab22a, particularly focusing on the commandeered Rab22a-associated endosomal maturation and endosomal cargo recycling, in addition to the extensively investigated oncogenic role of Rab22a.     

Effect of inhibiting vacuolar acidification on insulin signaling in hepatocytes

Previous studies have shown that the endosomal apparatus plays an important role in insulin signaling. Inhibition of endosomal acidification leads to a decrease in insulin-insulin receptor kinase (IRK) dissociation and insulin degradation. Thus, vacuolar pH could function as a modulator of insulin signaling in endosomes. In the present study we show that in primary hepatocytes pretreated with bafilomycin, there is an inhibition of vacuolar acidification. Incubation of these cells with insulin was followed by an augmentation of IRK activity but an inhibition of phosphatidylinositol 3-kinase/Akt activity and a decrease in insulin-induced DNA and glycogen synthesis. Bafilomycin treatment inhibited IRK recycling to the plasma membrane without affecting IRK internalization. Impaired IRK recycling correlated with a decrease in insulin signaling. We suggest that inhibiting vacuolar acidification sequesters activated IRKs in an intracellular compartment(s) where signaling is inhibited. This implies that endosomal receptor trafficking plays a role in regulating signal transduction.     

A Role for Cargo in Arf-dependent Adaptor Recruitment

Membrane traffic requires the specific concentration of protein cargos and exclusion of other proteins into nascent carriers. Critical components of this selectivity are the protein adaptors that bind to short, linear motifs in the cytoplasmic tails of transmembrane protein cargos and sequester them into nascent carriers. The recruitment of the adaptors is mediated by activated Arf GTPases, and the Arf-adaptor complexes mark sites of carrier formation. However, the nature of the signal(s) that initiates carrier biogenesis remains unknown. We examined the specificity and initial sites of recruitment of Arf-dependent adaptors (AP-1 and GGAs) in response to the Golgi or endosomal localization of specific cargo proteins (furin, mannose-6-phosphate receptor (M6PR), and M6PR lacking a C-terminal domain M6PRΔC). We find that cargo promotes the recruitment of specific adaptors, suggesting that it is part of an upstream signaling event. Cargos do not promote adaptor recruitment to all compartments in which they reside, and thus additional factors regulate the cargo''s ability to promote Arf activation and adaptor recruitment. We document that within a given compartment different cargos recruit different adaptors, suggesting that there is little or no free, activated Arf at the membrane and that Arf activation is spatially and temporally coupled to the cargo and the adaptor. Using temperature block, brefeldin A, and recovery from each, we found that the cytoplasmic tail of M6PR causes the recruitment of AP-1 and GGAs to recycling endosomes and not at the Golgi, as predicted by steady state staining profiles. These results are discussed with respect to the generation of novel models for cargo-dependent regulation of membrane traffic.     

Recycling of cell surface membrane proteins from yeast endosomes is regulated by ubiquitinated Ist1

Upon internalization, many surface membrane proteins are recycled back to the plasma membrane. Although these endosomal trafficking pathways control surface protein activity, the precise regulatory features and division of labor between interconnected pathways are poorly defined. In yeast, we show recycling back to the surface occurs through distinct pathways. In addition to retrograde recycling pathways via the late Golgi, used by synaptobrevins and driven by cargo ubiquitination, we find nutrient transporter recycling bypasses the Golgi in a pathway driven by cargo deubiquitination. Nutrient transporters rapidly internalize to, and recycle from, endosomes marked by the ESCRT-III associated factor Ist1. This compartment serves as both “early” and “recycling” endosome. We show Ist1 is ubiquitinated and that this is required for proper endosomal recruitment and cargo recycling to the surface. Additionally, the essential ATPase Cdc48 and its adaptor Npl4 are required for recycling, potentially through regulation of ubiquitinated Ist1. This collectively suggests mechanistic features of recycling from endosomes to the plasma membrane are conserved.     

The cell fate determinant numb interacts with EHD/Rme-1 family proteins and has a role in endocytic recycling

The adaptor protein Numb is necessary for the cell fate specification of progenitor cells in the Drosophila nervous system. Numb is evolutionarily conserved and previous studies have provided evidence for a similar functional role during mammalian development. The Numb protein has multiple protein-protein interaction regions including a phosphotyrosine binding (PTB) domain and a carboxy-terminal domain that contains conserved interaction motifs including an EH (Eps15 Homology) domain binding motif and alpha-adaptin binding site. In this study we identify the EHD/Rme-1/Pincher family of endocytic proteins as Numb interacting partners in mammals and Drosophila. The EHD/Rme-1 proteins function in recycling of plasma membrane receptors internalized by both clathrin-mediated endocytosis and a clathrin-independent pathway regulated by ADP ribosylation factor 6 (Arf6). Here we report that Numb colocalizes with endogenous EHD4/Pincher and Arf6 and that Arf6 mutants alter Numb subcellular localization. In addition, we present evidence that Numb has a novel function in endosomal recycling and intracellular trafficking of receptors.     

Rab4 affects both recycling and degradative endosomal trafficking

The small GTPases Rab4, Rab5 and Rab7 are endosomal proteins which play important roles in the regulation of various stages of endosomal trafficking. Rab4 and Rab5 have both been localized to early endosomes and have been shown to control recycling and endosomal fusion, respectively. Rab7, a marker of the late endosomal compartment, is involved in the regulation of the late endocytic pathway. Here, we compare the role of Rab4, Rab5 and Rab7 in early and late endosomal trafficking in HeLa cells monitoring ligand uptake, recycling and degradation. Expression of the Rab4 dominant negative mutant (Rab4AS22N) leads to a significant reduction in both recycling and degradation while, as expected, Rab7 mutants exclusively affect epidermal growth factor (EGF) and low density lipoprotein degradation. As also expected, expression of the dominant negative Rab5 mutant perturbs internalization kinetics and affects both recycling and degradation. Expression of Rab4WT and dominant positive mutant (Rab4AQ67L) changes dramatically the morphology of the transferrin compartment leading to the formation of membrane tubules. These transferrin positive tubules display swellings (varicosities) some of which are positive for early endosomal antigen-1 and contain EGF. We propose that the Rab4GTPase is important for the function of the early sorting endosomal compartment, affecting trafficking along both recycling and degradative pathways.