Molecular mechanism and physiological functions of clathrin-mediated endocytosis.

Clathrin-mediated endocytosis is the endocytic portal into cells through which cargo is packaged into vesicles with the aid of a clathrin coat. It is fundamental to neurotransmission, signal transduction and the regulation of many plasma membrane activities and is thus essential to higher eukaryotic life. Morphological stages of vesicle formation are mirrored by progression through various protein modules (complexes). The process involves the formation of a putative FCH domain only (FCHO) initiation complex, which matures through adaptor protein 2 (AP2)-dependent cargo selection, and subsequent coat building, dynamin-mediated scission and finally auxilin- and heat shock cognate 70 (HSC70)-dependent uncoating. Some modules can be used in other pathways, and additions or substitutions confer cell specificity and adaptability.

Adaptor protein2 (AP2) orchestrates CXCR2-mediated cell migration.

The chemokine receptor CXCR2 is vital for inflammation, wound healing, angiogenesis, cancer progression and metastasis. Adaptor protein 2 (AP2), a clathrin binding heterotetrameric protein comprised of α, ß2, µ2 and σ2 subunits, facilitates clathrin-mediated endocytosis. Mutation of the LLKIL motif in the CXCR2 carboxyl-terminal domain (CTD) results in loss of AP2 binding to the receptor and loss of ligand-mediated receptor internalization and chemotaxis. AP2 knockdown also results in diminished ligand-mediated CXCR2 internalization, polarization and chemotaxis. Using knockdown/rescue approaches with AP2-µ2 mutants, the binding domains were characterized in reference to CXCR2 internalization and chemotaxis. When in an open conformation, µ2 Patch 1 and Patch 2 domains bind tightly to membrane PIP2 phospholipids. When AP2-µ2, is replaced with µ2 mutated in Patch 1 and/or Patch 2 domains, ligand-mediated receptor binding and internalization are not lost. However, chemotaxis requires AP2-µ2 Patch 1, but not Patch 2. AP2-σ2 has been demonstrated to bind dileucine motifs to facilitate internalization. Expression of AP2-σ2 V88D and V98S dominant negative mutants resulted in loss of CXCR2 mediated chemotaxis. Thus, AP2 binding to both membrane phosphatidylinositol phospholipids and dileucine motifs is crucial for directional migration or chemotaxis. Moreover, AP2-mediated receptor internalization can be dissociated from AP2-mediated chemotaxis.

TBC1D5 and the AP2 complex regulate ATG9 trafficking and initiation of autophagy.

The RabGAP protein TBC1D5 controls cellular endomembrane trafficking processes and binds the retromer subunit VPS29 and the ubiquitin-like protein ATG8 (LC3). Here, we describe that TBC1D5 also associates with ATG9 and the active ULK1 complex during autophagy. Moreover, ATG9 and TBC1D5 interact with clathrin and the AP2 complex. Depletion of TBC1D5 leads to missorting of ATG9 to late endosomes upon activation of autophagy, whereas inhibition of clathrin-mediated endocytosis or AP2 depletion alters ATG9 trafficking and its association with TBC1D5. Taken together, our data show that TBC1D5 and the AP2 complex are important novel regulators of the rerouting of ATG9-containing vesicular carriers toward sites of autophagosome formation.

New Insights to Clathrin and Adaptor Protein 2 for the Design and Development of Therapeutic Strategies.

The Amyloid Precursor Protein (APP) has been extensively studied for its role as the precursor of the ß-amyloid protein (Aß) in Alzheimer's disease (AD). However, our understanding of the normal function of APP is still patchy. Emerging evidence indicates that a dysfunction in APP trafficking and degradation can be responsible for neuronal deficits and progressive degeneration in humans. We recently reported that the Y682 mutation in the 682YENPTY687 domain of APP affects its binding to specific adaptor proteins and leads to its anomalous trafficking, to defects in the autophagy machinery and to neuronal degeneration. In order to identify adaptors that influence APP function, we performed pull-down experiments followed by quantitative mass spectrometry (MS) on hippocampal tissue extracts of three month-old mice incubated with either the 682YENPTY687 peptide, its mutated form, 682GENPTY687 or its phosphorylated form, 682pYENPTY687. Our experiments resulted in the identification of two proteins involved in APP internalization and trafficking: Clathrin heavy chain (hc) and its Adaptor Protein 2 (AP-2). Overall our results consolidate and refine the importance of Y682 in APP normal functions from an animal model of premature aging and dementia. Additionally, they open the perspective to consider Clathrin hc and AP-2 as potential targets for the design and development of new therapeutic strategies.

Otoferlin couples to clathrin-mediated endocytosis in mature cochlear inner hair cells.

The encoding of auditory information with indefatigable precision requires efficient resupply of vesicles at inner hair cell (IHC) ribbon synapses. Otoferlin, a transmembrane protein responsible for deafness in DFNB9 families, has been postulated to act as a calcium sensor for exocytosis as well as to be involved in rapid vesicle replenishment of IHCs. However, the molecular basis of vesicle recycling in IHCs is largely unknown. In the present study, we used high-resolution liquid chromatography coupled with mass spectrometry to copurify otoferlin interaction partners in the mammalian cochlea. We identified multiple subunits of the adaptor protein complex AP-2 (CLAP), an essential component of clathrin-mediated endocytosis, as binding partners of otoferlin in rats and mice. The interaction between otoferlin and AP-2 was confirmed by coimmunoprecipitation. We also found that AP-2 interacts with myosin VI, another otoferlin binding partner important for clathrin-mediated endocytosis (CME). The expression of AP-2 in IHCs was verified by reverse transcription PCR. Confocal microscopy experiments revealed that the expression of AP-2 and its colocalization with otoferlin is confined to mature IHCs. When CME was inhibited by blocking dynamin action, real-time changes in membrane capacitance showed impaired synaptic vesicle replenishment in mature but not immature IHCs. We suggest that an otoferlin-AP-2 interaction drives Ca(2+)- and stimulus-dependent compensating CME in mature IHCs.

The endocytosis of cellulose synthase in Arabidopsis is dependent on µ2, a clathrin-mediated endocytosis adaptin.

Clathrin-mediated endocytosis (CME) is the best-characterized type of endocytosis in eukaryotic cells. Plants appear to possess all of the molecular components necessary to carry out CME; however, functional characterization of the components is still in its infancy. A yeast two-hybrid screen identified µ2 as a putative interaction partner of CELLULOSE SYNTHASE6 (CESA6). Arabidopsis (Arabidopsis thaliana) µ2 is homologous to the medium subunit 2 of the mammalian ADAPTOR PROTEIN COMPLEX2 (AP2). In mammals, the AP2 complex acts as the central hub of CME by docking to the plasma membrane while concomitantly recruiting cargo proteins, clathrin triskelia, and accessory proteins to the sites of endocytosis. We confirmed that µ2 interacts with multiple CESA proteins through the µ-homology domain of µ2, which is involved in specific interactions with endocytic cargo proteins in mammals. Consistent with its role in mediating the endocytosis of cargos at the plasma membrane, µ2-YELLOW FLUORESCENT PROTEIN localized to transient foci at the plasma membrane, and loss of µ2 resulted in defects in bulk endocytosis. Furthermore, loss of µ2 led to increased accumulation of YELLOW FLUORESCENT PROTEIN-CESA6 particles at the plasma membrane. Our results suggest that CESA represents a new class of CME cargo proteins and that plant cells might regulate cellulose synthesis by controlling the abundance of active CESA complexes at the plasma membrane through CME.

C. elegans AP-2 and retromer control Wnt signaling by regulating mig-14/Wntless.

While endocytosis can regulate morphogen distribution, its precise role in shaping these gradients is unclear. Even more enigmatic is the role of retromer, a complex that shuttles proteins between endosomes and the Golgi apparatus, in Wnt gradient formation. Here we report that DPY-23, the C. elegans mu subunit of the clathrin adaptor AP-2 that mediates the endocytosis of membrane proteins, regulates Wnt function. dpy-23 mutants display Wnt phenotypes, including defects in neuronal migration, neuronal polarity, and asymmetric cell division. DPY-23 acts in Wnt-expressing cells to promote these processes. MIG-14, the C. elegans homolog of the Wnt-secretion factor Wntless, also acts in these cells to control Wnt function. In dpy-23 mutants, MIG-14 accumulates at or near the plasma membrane. By contrast, MIG-14 accumulates in intracellular compartments in retromer mutants. Based on our observations, we propose that intracellular trafficking of MIG-14 by AP-2 and retromer plays an important role in Wnt secretion.

AP2 adaptor complex mediates bile salt export pump internalization and modulates its hepatocanalicular expression and transport function.

UNLABELLED: The bile salt export pump (BSEP) mediates the biliary excretion of bile salts and its dysfunction induces intrahepatic cholestasis. Reduced canalicular expression of BSEP resulting from the promotion of its internalization is one of the causes of this disease state. However, the molecular mechanism underlying BSEP internalization from the canalicular membrane (CM) remains unknown. We have shown previously that 4-phenylbutyrate (4PBA), a drug used for ornithine transcarbamylase deficiency (OTCD), inhibited internalization and subsequent degradation of cell-surface-resident BSEP. The current study found that 4PBA treatment decreased significantly the expression of α- and µ2-adaptin, both of which are subunits of the AP2 adaptor complex (AP2) that mediates clathrin-dependent endocytosis, in liver specimens from rats and patients with OTCD, and that BSEP has potential AP2 recognition motifs in its cytosolic region. Based on this, the role of AP2 in BSEP internalization was explored further. In vitro analysis with 3×FLAG-human BSEP-expressing HeLa cells and human sandwich-culture hepatocytes indicates that the impairment of AP2 function by RNA interference targeting of α-adaptin inhibits BSEP internalization from the plasma membrane and increases its cell-surface expression and transport function. Studies using immunostaining, coimmunoprecipitation, glutathione S-transferase pulldown assay, and time-lapse imaging show that AP2 interacts with BSEP at the CM through a tyrosine motif at the carboxyl terminus of BSEP and mediates BSEP internalization from the CM of hepatocytes. CONCLUSION: AP2 mediates the internalization and subsequent degradation of CM-resident BSEP through direct interaction with BSEP and thereby modulates the canalicular expression and transport function of BSEP. This information should be useful for understanding the pathogenesis of severe liver diseases associated with intrahepatic cholestasis.

Ubiquitination differentially regulates clathrin-dependent internalization of protease-activated receptor-1.

Protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is irreversibly activated by proteolysis. Consequently, PAR1 trafficking is critical for the fidelity of thrombin signaling. PAR1 displays constitutive and agonist-induced internalization, which are clathrin and dynamin dependent but are independent of arrestins. The clathrin adaptor AP2 (adaptor protein complex-2) is critical for constitutive but not for activated PAR1 internalization. In this study, we show that ubiquitination negatively regulates PAR1 constitutive internalization and specifies a distinct clathrin adaptor requirement for activated receptor internalization. PAR1 is basally ubiquitinated and deubiquitinated after activation. A PAR1 lysineless mutant signaled normally but was not ubiquitinated. Constitutive internalization of ubiquitin (Ub)-deficient PAR1 was markedly increased and inhibited by the fusion of Ub to the cytoplasmic tail. Ub-deficient PAR1 constitutive internalization was AP2 dependent like the wild-type receptor. However, unlike wild-type PAR1, AP2 was required for the internalization of activated Ub-deficient receptor, suggesting that the internalization of ubiquitinated PAR1 requires different endocytic machinery. These studies reveal a novel function for ubiquitination in the regulation of GPCR internalization.

Eps15 and Epsin1 are crucial for enteropathogenic Escherichia coli pedestal formation despite the absence of adaptor protein 2.

Enteropathogenic Escherichia coli (EPEC) are primarily extracellular pathogens that generate actin-rich structures known as pedestals during their pathogenesis. Surprising evidence has demonstrated that despite maintaining an extracellular location, EPEC require the endocytic protein, clathrin, for pedestal formation. To evaluate the strategies EPEC use to usurp endocytic machinery, we investigated the roles of a number of clathrin-coated pits components, adaptor protein 2 (AP-2), Eps15 and epsin1, during EPEC infections. We demonstrated that in conjunction with clathrin, pedestal formation also required the recruitment of Eps15 and epsin1 but not AP-2. Because AP-2 orchestrates the recruitment of clathrin, Eps15, and epsin1, as well as other adaptors, during assembly of clathrin-coated pits at the plasma membrane, our findings reveal a novel internalization subversion strategy employed by EPEC. These results further emphasize the recent paradigm that endocytic proteins are important for EPEC-mediated disease.

Viral factors involved in adapter-related protein complex 2 alpha 1 subunit-mediated regulation of human immunodeficiency virus type 1 replication.

The presence of siRNA against adapter-related protein complex 2 alpha 1 subunit (AP2alpha) enhances human immunodeficiency virus type 1 (HIV-1) replication by up-regulating nuclear transport of viral genome. In this report, we examined possible viral factors involved in AP2alpha-mediated regulation of HIV-1 replication, namely, Gag matrix protein (MA), integrase (IN) and Vpr. Replication of mutant viruses lacking the nucleophilic property of one of these viral proteins was significantly enhanced by treating cells with AP2alpha siRNA, indicating that Gag MA, IN or Vpr is not specifically involved in AP2alpha-mediated enhancement of viral replication. In contrast, AP2alpha siRNA showed no effect on the level of gene transduction mediated by HIV-1-derived lentiviral vector (LV). Although virus-like LV particle and parental HIV-1 particle are composed of almost equivalent viral structural proteins, LV particles lack three accessory proteins, Vif, Vpr and Vpu, and a large portion of the HIV-1 genome. Vif, Vpr and Vpu were dispensable for AP2alpha siRNA-mediated enhancement of HIV-1 replication, indicating that a particular part of the HIV-1 genomic fragment deleted in the LV genome might be required for the enhancing effect of AP2alpha siRNA on viral replication. Taken together, these results suggest that an as yet undetermined gene fragment of the HIV-1 genome is involved in AP2alpha-mediated regulation of HIV-1 replication.

Synaptic vesicle recycling at CNS snapses without AP-2.

Synaptic vesicles (SVs) are composed of approximately 10 types of transmembrane proteins that must be recycled after exocytosis of neurotransmitter. The mechanisms for resorting these proteins into synaptic vesicles once incorporated into the plasma membrane after exocytosis are poorly understood. The adaptor complex AP-2 is the major clathrin-associated adaptor for cargo recognition at the plasma membrane. Here, we have investigated its role in synaptic vesicle endocytosis. shRNA-mediated knockdown of the AP-2 complex results in an approximately 96% reduction of this protein complex in primary neurons. We used simultaneous expression of shRNA and pHluorin-tagged vesicle components to show that the absence of AP-2 significantly slows but does not prevent the endocytosis of four of the major synaptic vesicle transmembrane proteins. We show that in the absence of AP-2, the AP-1 adaptor complex appears to functionally substitute for AP-2 but results in complex internalization kinetics that are now sensitive to the guanine-nucleotide exchange factor for ADP-ribosylation factor GTPase (ARF-GEF) inhibitor brefeldin-A (BFA). Simultaneous removal of both AP-2 and AP-1 prevents this compensatory substitution and results in slowed but functional endocytosis. These results demonstrate that in the absence of AP-2, SV proteins still become endocytosed, and synaptic vesicle recycling remains operational.

A distributed set of interactions controls mu2 functionality in the role of AP-2 as a sorting adaptor in synaptic vesicle endocytosis.

The mechanisms of how, following exocytosis, the approximately nine types of synaptic vesicle (SV) transmembrane proteins are accurately resorted to form SVs are poorly understood. The time course of SV endocytosis is very sensitive to perturbations in clathrin and dynamin, supporting the model that SV endocytosis occurs through a clathrin-mediated pathway. We recently demonstrated that removal of the clathrin adaptor protein AP-2, the key protein thought to coordinate cargo selection into clathrin-coated pits, results in a significant impairment in endocytosis kinetics. Endocytosis, however, still proceeds in the absence of AP-2, bringing into question the role of AP-2 in cargo sorting in this process. Using quantitative endocytosis assays at nerve terminals, we examined how endocytosis depends on the integrity of mu2 function. Our experiments indicate that no single perturbation in mu2 prevents restoration of endocytic function when mutated mu2 replaces native mu2, whereas introduction of multiple distributed mutations significantly impairs endocytosis. We also examined whether the presence of AP-2 is important for the functionality of the previously identified endocytic motif in an SV cargo protein, the dileucine motif in vGlut-1. These data show that while mutations in the dileucine motif slow the retrieval of vGlut-1, they only do so in the presence of AP-2. These data thus indicate that AP-2 plays a role in cargo selection but that no single aspect of mu2 function is critical, implying that a more distributed network of interactions supports AP-2 function in SV endocytosis.

Membrane targeting of endocytic adaptors: cargo and lipid do it together.

The adaptor complex AP-2 plays an important role in cargo selection and clathrin lattice formation during clathrin-mediated endocytosis. In a recent issue of Molecular Cell, Honing et al. demonstrate that high-affinity AP-2 membrane association is achieved through a combination of low-affinity interactions with membrane phosphoinositides and cargo proteins, regulated by phosphorylation.

Human immunodeficiency virus type-1 gag and host vesicular trafficking pathways.

The Gag protein of HIV-1 directs the particle assembly process. Gag recruits components of the cellular vesicular trafficking machinery in order to traverse the cytoplasm of the cell and reach the particle assembly site. The plasma membrane is the primary site of particle assembly in most cell types, while in macrophages an unusual intracellular membrane-bound compartment bearing markers of late endosomes and the plasma membrane is the predominant assembly site. Plasma membrane specificity of assembly may be directed by components of lipid rafts and the cytoplasmic leaflet component PI(4,5)P(2). Recent work has highlighted the role of adaptor protein complexes, protein sorting and recycling pathways, components of the multivesicular body, and cellular motor proteins in facilitating HIV assembly and budding. This review presents an overview of the relevant vesicular trafficking pathways and describes the individual components implicated in interactions with Gag.

Imaging poliovirus entry in live cells.

Viruses initiate infection by transferring their genetic material across a cellular membrane and into the appropriate compartment of the cell. The mechanisms by which animal viruses, especially nonenveloped viruses, deliver their genomes are only poorly understood. This is due in part to technical difficulties involved in direct visualization of viral gene delivery and to uncertainties in distinguishing productive and nonproductive pathways caused by the high particle-to-plaque forming unit ratio of most animal viruses. Here, we combine an imaging assay that simultaneously tracks the viral capsid and genome in live cells with an infectivity-based assay for RNA release to characterize the early events in the poliovirus (PV) infection. Effects on RNA genome delivery from inhibitors of cell trafficking pathways were probed systematically by both methods. Surprisingly, we observe that genome release by PV is highly efficient and rapid, and thus does not limit the overall infectivity or the infection rate. The results define a pathway in which PV binds to receptors on the cell surface and enters the cell by a clathrin-, caveolin-, flotillin-, and microtubule-independent, but tyrosine kinase- and actin-dependent, endocytic mechanism. Immediately after the internalization of the virus particle, genome release takes place from vesicles or tightly sealed membrane invaginations located within 100-200 nm of the plasma membrane. These results settle a long-lasting debate of whether PV directly breaks the plasma membrane barrier or relies on endocytosis to deliver its genome into the cell. We expect this imaging assay to be broadly applicable to the investigation of entry mechanisms for nonenveloped viruses.

Phosphatidic acid increases Notch signalling by affecting Sanpodo trafficking during Drosophila sensory organ development.

Organ cell diversity depends on binary cell-fate decisions mediated by the Notch signalling pathway during development and tissue homeostasis. A clear example is the series of binary cell-fate decisions that take place during asymmetric cell divisions that give rise to the sensory organs of Drosophila melanogaster. The regulated trafficking of Sanpodo, a transmembrane protein that potentiates receptor activity, plays a pivotal role in this process. Membrane lipids can regulate many signalling pathways by affecting receptor and ligand trafficking. It remains unknown, however, whether phosphatidic acid regulates Notch-mediated binary cell-fate decisions during asymmetric cell divisions, and what are the cellular mechanisms involved. Here we show that increased phosphatidic acid derived from Phospholipase D leads to defects in binary cell-fate decisions that are compatible with ectopic Notch activation in precursor cells, where it is normally inactive. Null mutants of numb or the α-subunit of Adaptor Protein complex-2 enhance dominantly this phenotype while removing a copy of Notch or sanpodo suppresses it. In vivo analyses show that Sanpodo localization decreases at acidic compartments, associated with increased internalization of Notch. We propose that Phospholipase D-derived phosphatidic acid promotes ectopic Notch signalling by increasing receptor endocytosis and inhibiting Sanpodo trafficking towards acidic endosomes.

Cdk5 regulates the phosphorylation of tyrosine 1472 NR2B and the surface expression of NMDA receptors.

NMDA receptors (NMDARs) are a major class of ionotropic glutamate receptors that can undergo activity-dependent changes in surface expression. Clathrin-mediated endocytosis is a mechanism by which the surface expression of NR2B-containing NMDA receptors is regulated. The C terminus of the NMDA receptor subunit NR2B contains the internalization motif YEKL, which is the binding site for the clathrin adaptor AP-2. The tyrosine (Y1472) within the YEKL motif is phosphorylated by the Src family of kinases and this phosphorylation inhibits the binding of AP-2 and promotes surface expression of NMDA receptors. Cdk5 is a serine/threonine kinase that has been implicated in synaptic plasticity, learning, and memory. Here we demonstrate that inhibition of Cdk5 results in increased phosphorylation of Y1472 NR2B at synapses and decreased binding of NR2B to beta2-adaptin, a subunit of AP-2, thus blocking the activity-dependent endocytosis of NMDA receptors. Furthermore, we show that inhibition of Cdk5 increases the binding of Src to postsynaptic density-95 (PSD-95), and that expression of PSD-95 facilitates the phosphorylation of Y1472 NR2B by Src. Together, these results suggest a model in which inhibition of Cdk5 increases the binding of Src to PSD-95 and the phosphorylation of Y1472 NR2B by Src, which results in decreased binding of NR2B to AP-2, and NR2B/NMDAR endocytosis. This study provides a novel molecular mechanism for the regulation of the surface expression of NR2B-containing NMDA receptors and gives insight into the Cdk5-dependent regulation of synaptic plasticity.

AP-2 makes room for rivals.

Clathrin and the adaptor protein complex (AP-2) constitute the major coat components of clathrin-coated vesicles. In the September issues of the Journal of Cell Biology and the Journal of Biological Chemistry, three reports reveal that AP-2, while essential for internalization of transferrin, is not essential for internalization of EGF. These novel data suggest the intriguing possibility that the major role of AP-2 is in cargo recruitment, and not in assembly of functionally active clathrin-coated pits.

Role of the AP2 beta-appendage hub in recruiting partners for clathrin-coated vesicle assembly.

Adaptor protein complex 2 alpha and beta-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of beta-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the beta-appendage (the "top" and "side" sites) that bind motifs distinct from those previously identified on the alpha-appendage. We solved the structure of the beta-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor beta-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the beta-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability ("matricity"). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as beta-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.