Endocytic Adaptor Protein Research Articles

Adaptor Protein 2 (AP-2) complex is essential for functional axogenesis in hippocampal neurons

The complexity and diversity of a neural network requires regulated elongation and branching of axons, as well as the formation of synapses between neurons. In the present study we explore the role of AP-2, a key endocytic adaptor protein complex, in the development of rat hippocampal neurons. We found that the loss of AP-2 during the early stage of development resulted in impaired axon extension and failed maturation of the axon initial segment (AIS). Normally the AIS performs two tasks in concert, stabilizing neural polarity and generating action potentials. In AP-2 silenced axons polarity is established, however there is a failure to establish action potential firing. Consequently, this impairs activity-driven Ca2+ influx and exocytosis at nerve terminals. In contrast, removal of AP-2 from older neurons does not impair axonal growth or signaling and synaptic function. Our data reveal that AP-2 has important roles in functional axogenesis by proper extension of axon as well as the formation of AIS during the early step of neurodevelopment.

Adaptor proteins NUMB and NUMBL promote cell cycle withdrawal by targeting ERBB2 for degradation.

Failure of trabecular myocytes to undergo appropriate cell cycle withdrawal leads to ventricular noncompaction and heart failure. Signaling of growth factor receptor ERBB2 is critical for myocyte proliferation and trabeculation. However, the mechanisms underlying appropriate downregulation of trabecular ERBB2 signaling are little understood. Here, we have found that the endocytic adaptor proteins NUMB and NUMBL were required for downregulation of ERBB2 signaling in maturing trabeculae. Loss of NUMB and NUMBL resulted in a partial block of late endosome formation, resulting in sustained ERBB2 signaling and STAT5 activation. Unexpectedly, activated STAT5 overrode Hippo-mediated inhibition and drove YAP1 to the nucleus. Consequent aberrant cardiomyocyte proliferation resulted in ventricular noncompaction that was markedly rescued by heterozygous loss of function of either ERBB2 or YAP1. Further investigations revealed that NUMB and NUMBL interacted with small GTPase Rab7 to transition ERBB2 from early to late endosome for degradation. Our studies provide insight into mechanisms by which NUMB and NUMBL promote cardiomyocyte cell cycle withdrawal and highlight previously unsuspected connections between pathways that are important for cardiomyocyte cell cycle reentry, with relevance to ventricular noncompaction cardiomyopathy and regenerative medicine.

Mimetic peptide of ubiquitin-interacting motif of epsin as a cancer therapeutic-perspective in brain tumor therapy through regulating VEGFR2 signaling.

Epsins, endocytic adaptor proteins required for internalization of ubiquitylated receptors, are generally upregulated in human cancers. It has been characterized that mice deficient of epsins in the endothelium inhibit tumor growth by dysregulating vascular endothelial growth factor receptor-2 (VEGFR2) signaling and non-productive tumor angiogenesis. Binding of the epsin ubiquitin (Ub)-interacting motif (UIM) with ubiquitylated VEGFR2 is a critical mechanism for epsin-dependent VEGFR2 endocytosis and degradation, indicative of epsin UIM as a potential therapeutic target. A Computer Assisted Drug Design approach was utilized to create the UIM mimetic peptides for the functional competition of epsin binding sites in ubiquitylated VEGFR2 in vivo. Specifically targeting VEGFR2 in the tumor vasculature, the chemically synthesized chimeric UIM peptide, UPI, causes non-functional tumor angiogenesis, retards tumor growth, and increases survival rates in several tumor models. The authors showed that UPI binds ubiquitylated VEGFR2 to form a supercomplex in an Ub-dependent fashion. Collectively, the UPI targeting strategy offers a potentially novel treatment for cancer patients who are resistant to current anti-angiogenic therapies. In this review, the authors outline the main points of this research specifically as a potential application for glioma tumor therapy.

Msx1-Positive Progenitors in the Retinal Ciliary Margin Give Rise to Both Neural and Non-neural Progenies in Mammals

In lower vertebrates, stem/progenitor cells located in a peripheral domain of the retina, called the ciliary margin zone (CMZ), cooperate with retinal domain progenitors to build the mature neural retina. In mammals, it is believed that the CMZ lacks neurogenic potential and that the retina develops from one pool of multipotent retinal progenitor cells (RPCs). Here we identify a population of Msx1-expressing progenitors in the mouse CMZ that is both molecularly and functionally distinct from RPCs. Using genetic lineage tracing, we report that Msx1 progenitors have unique developmental properties compared with RPCs. Msx1 lineages contain both neural retina and non-neural ciliary epithelial progenies and overall generate fewer photoreceptors than classical RPC lineages. Furthermore, we show that the endocytic adaptor protein Numb regulates the balance between neural and non-neural fates in Msx1 progenitors. These results uncover a population of CMZ progenitors, distinct from classical RPCs, that also contributes to mammalian retinogenesis.

Internalization of Heterologous Sugar Transporters by Endogenous α-Arrestins in the Yeast Saccharomyces cerevisiae.

ABSTRACTWhen expressed in Saccharomyces cerevisiae using either of two constitutive yeast promoters (PGK1prom and CCW12prom), the transporters CDT-1 and CDT-2 from the filamentous fungus Neurospora crassa are able to catalyze, respectively, active transport and facilitated diffusion of cellobiose (and, for CDT-2, also xylan and its derivatives). In S. cerevisiae, endogenous permeases are removed from the plasma membrane by clathrin-mediated endocytosis and are marked for internalization through ubiquitinylation catalyzed by Rsp5, a HECT class ubiquitin:protein ligase (E3). Recruitment of Rsp5 to specific targets is mediated by a 14-member family of endocytic adaptor proteins, termed α-arrestins. Here we demonstrate that CDT-1 and CDT-2 are subject to α-arrestin-mediated endocytosis, that four α-arrestins (Rod1, Rog3, Aly1, and Aly2) are primarily responsible for this internalization, that the presence of the transport substrate promotes transporter endocytosis, and that, at least for CDT-2, residues located in its C-terminal cytosolic domain are necessary for its efficient endocytosis. Both α-arrestin-deficient cells expressing CDT-2 and otherwise wild-type cells expressing CDT-2 mutants unresponsive to α-arrestin-driven internalization exhibit an increased level of plasma membrane-localized transporter compared to that of wild-type cells, and they grow, utilize the transport substrate, and generate ethanol anaerobically better than control cells.IMPORTANCE Ethanolic fermentation of the breakdown products of plant biomass by budding yeast Saccharomyces cerevisiae remains an attractive biofuel source. To achieve this end, genes for heterologous sugar transporters and the requisite enzyme(s) for subsequent metabolism have been successfully expressed in this yeast. For one of the heterologous transporters examined in this study, we found that the amount of this protein residing in the plasma membrane was the rate-limiting factor for utilization of the cognate carbon source (cellobiose) and its conversion to ethanol.

Disabled-2 Determines Commitment of a Pre-adipocyte Population in Juvenile Mice.

Disabled-2 (Dab2) is a widely expressed clathrin binding endocytic adaptor protein and known for the endocytosis of the low-density lipoprotein (LDL) family receptors. Dab2 also modulates endosomal Ras/MAPK (Erk1/2) activity by regulating the disassembly of Grb2/Sos1 complexes associated with clathrin-coated vesicles. We found that the most prominent phenotype of Dab2 knockout mice was their striking lean body composition under a high fat and high caloric diet, although the weight of the mutant mice was indistinguishable from wild-type littermates on a regular chow. The remarkable difference in resistance to high caloric diet-induced weight gain of the dab2-deleted mice was presented only in juvenile but not in mature mice. Investigation using Dab2-deficient embryonic fibroblasts and mesenchymal stromal cells indicated that Dab2 promoted adipogenic differentiation by modulation of MAPK (Erk1/2) activity, which otherwise suppresses adipogenesis through the phosphorylation of PPARγ. The results suggest that Dab2 is required for the excessive calorie-induced differentiation of an adipocyte progenitor cell population that is present in juvenile but depleted in mature animals. The finding provides evidence for a limited pre-adipocyte population in juvenile mammals and the requirement of Dab2 in the regulation of Ras/MAPK signal in the commitment of the precursor cells to adipose tissues.

Disabled-2 is a negative immune regulator of lipopolysaccharide-stimulated Toll-like receptor 4 internalization and signaling.

Toll-like receptor 4 (TLR4) plays a pivotal role in the host response to lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria. Here, we elucidated whether the endocytic adaptor protein Disabled-2 (Dab2), which is abundantly expressed in macrophages, plays a role in LPS-stimulated TLR4 signaling and trafficking. Molecular analysis and transcriptome profiling of RAW264.7 macrophage-like cells expressing short-hairpin RNA of Dab2 revealed that Dab2 regulated the TLR4/TRIF pathway upon LPS stimulation. Knockdown of Dab2 augmented TRIF-dependent interferon regulatory factor 3 activation and the expression of subsets of inflammatory cytokines and interferon-inducible genes. Dab2 acted as a clathrin sponge and sequestered clathrin from TLR4 in the resting stage of macrophages. Upon LPS stimulation, clathrin was released from Dab2 to facilitate endocytosis of TLR4 for triggering the TRIF-mediated pathway. Dab2 functions as a negative immune regulator of TLR4 endocytosis and signaling, supporting a novel role for a Dab2-associated regulatory circuit in controlling the inflammatory response of macrophages to endotoxin.

The polarity protein Par3 regulates APP trafficking and processing through the endocytic adaptor protein Numb

The processing of amyloid precursor protein (APP) into β-amyloid peptide (Aβ) is a key step in the pathogenesis of Alzheimer’s disease (AD), and trafficking dysregulations of APP and its secretases contribute significantly to altered APP processing. Here we show that the cell polarity protein Par3 plays an important role in APP processing and trafficking. We found that the expression of full length Par3 is significantly decreased in AD patients. Overexpression of Par3 promotes non-amyloidogenic APP processing, while depletion of Par3 induces intracellular accumulation of Aβ. We further show that Par3 functions by regulating APP trafficking. Loss of Par3 decreases surface expression of APP by targeting APP to the late endosome/lysosome pathway. Finally, we show that the effects of Par3 are mediated through the endocytic adaptor protein Numb, and Par3 functions by interfering with the interaction between Numb and APP. Together, our studies show a novel role for Par3 in regulating APP processing and trafficking.

Regulation of Numb isoform expression by activated ERK signaling.

The endocytic adaptor protein Numb has a major role in development as an intrinsic regulator of cell fate determination and inhibitor of the Notch signaling pathway. In vertebrates, four protein isoforms of Numb are produced through alternative splicing (AS) of two cassette exons (exons 3 and 9). AS of coding exon 9 (E9) produces E9-included (p72/p71) and -excluded (p66/p65) protein products. Expression of Numb isoforms is developmentally regulated and E9-included products are expressed in progenitors, whereas E9-excluded isoforms are dominantly expressed in differentiated cells. Analyses of AS events in multiple cancers previously identified a switch in Numb transcript and protein expression from the E9-excluded to the E9-included isoform, suggesting that misregulation of the mechanisms that control E9 inclusion may have a role in tumorigenesis. Here we identify splicing factors ASF/SF2 and PTBP1 as regulators of E9 splicing and show that activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway promotes E9 inclusion in cancer cells. Our evidence supports a mechanism by which Numb AS is regulated in response to oncogenic signaling pathways, and contributes to activation of downstream pathways to promote tumorigenesis.

Chapter Nine - Cellular Roles of Beta-Arrestins as Substrates and Adaptors of Ubiquitination and Deubiquitination.

β-Arrestin1 and β-arrestin2 are homologous adaptor proteins that are ubiquitously expressed in mammalian cells. They belong to a four-member family of arrestins that regulate the vast family of seven-transmembrane receptors that couple to heterotrimeric G proteins (7TMRs or GPCRs), and that modulate 7TMR signal transduction. β-Arrestins were originally identified in the context of signal inhibition via the 7TMRs because they competed with and thereby blocked G protein coupling to 7TMRs. Currently, in addition to their role as desensitizers of signaling, β-arrestins are appreciated as multifunctional adaptors that mediate trafficking and signal transduction of not only 7TMRs, but a growing list of additional receptors, ion channels, and nonreceptor proteins. β-Arrestins' interactions with their multifarious partners are based on their dynamic conformational states rather than particular domain-domain interactions. β-Arrestins adopt activated conformations upon 7TMR association. In addition, β-arrestins undergo various posttranslational modifications that are choreographed by activated 7TMRs, including phosphorylation, ubiquitination, acetylation, nitrosylation, and SUMOylation. Ubiquitination of β-arrestins is critical for their high-affinity interaction with 7TMRs as well as with endocytic adaptor proteins and signaling kinases. β-Arrestins also function as critical adaptors for ubiquitination and deubiquitination of various cellular proteins, and thereby affect the longevity of signal transducers and the intensity of signal transmission.

Numb is required to prevent p53-dependent senescence following skeletal muscle injury

Regeneration relies on coordinated action of multiple cell types to reconstitute the damaged tissue. Here we inactivate the endocytic adaptor protein Numb in skeletal muscle stem cells prior to chronic or severe muscle injury in mice. We observe two types of senescence in regenerating muscle; a transient senescence in non-myogenic cells of control and Numb mutant mice that partly depends on INK4a/ARF activity, and a persistent senescence in myogenic cells lacking Numb. The senescence levels of Numb-deficient muscle is reduced to wild type levels by an anti-oxidant treatment or p53 ablation, resulting in functional rescue of the regenerative potential in Numb mutants. Ex vivo experiments suggest that Numb-deficient senescent cells recruit macrophages to sustain inflammation and drive fibrosis, two hallmarks of the impaired muscle regeneration in Numb mutants. These findings provide insights into previously reported developmental and oncogenic senescence that are also differentially regulated by p53.

Disruption of adaptor protein 2μ (AP-2μ) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing.

Active zones (AZs) of inner hair cells (IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. Here, we report that the endocytic adaptor protein 2μ (AP-2μ) is required for release site replenishment and hearing. We show that hair cell-specific disruption of AP-2μ slows IHC exocytosis immediately after fusion of the readily releasable pool of vesicles, despite normal abundance of membrane-proximal vesicles and intact endocytic membrane retrieval. Sound-driven postsynaptic spiking was reduced in a use-dependent manner, and the altered interspike interval statistics suggested a slowed reloading of release sites. Sustained strong stimulation led to accumulation of endosome-like vacuoles, fewer clathrin-coated endocytic intermediates, and vesicle depletion of the membrane-distal synaptic ribbon in AP-2μ-deficient IHCs, indicating a further role of AP-2μ in clathrin-dependent vesicle reformation on a timescale of many seconds. Finally, we show that AP-2 sorts its IHC-cargo otoferlin. We propose that binding of AP-2 to otoferlin facilitates replenishment of release sites, for example, via speeding AZ clearance of exocytosed material, in addition to a role of AP-2 in synaptic vesicle reformation.

Intrinsically disordered proteins drive membrane curvature.

Assembly of highly curved membrane structures is essential to cellular physiology. The prevailing view has been that proteins with curvature-promoting structural motifs, such as wedge-like amphipathic helices and crescent-shaped BAR domains, are required for bending membranes. Here we report that intrinsically disordered domains of the endocytic adaptor proteins, Epsin1 and AP180 are highly potent drivers of membrane curvature. This result is unexpected since intrinsically disordered domains lack a well-defined three-dimensional structure. However, in vitro measurements of membrane curvature and protein diffusivity demonstrate that the large hydrodynamic radii of these domains generate steric pressure that drives membrane bending. When disordered adaptor domains are expressed as transmembrane cargo in mammalian cells, they are excluded from clathrin-coated pits. We propose that a balance of steric pressure on the two surfaces of the membrane drives this exclusion. These results provide quantitative evidence for the influence of steric pressure on the content and assembly of curved cellular membrane structures.

Endophilin A2 Promotes TNBC Cell Invasion and Tumor Metastasis.

Triple-negative breast cancers (TNBCs) are highly aggressive cancers that lack targeted therapies. However, EGFR is frequently activated in a subset of TNBCs and represents a viable clinical target. Because the endocytic adaptor protein Endophilin A2 (SH3GL1/Endo II) has been implicated in EGFR internalization, we investigated Endo II expression and function in human TNBCs. Endo II expression was high in several TNBC cells compared with normal breast epithelial cells. Stable knockdown (KD) of Endo II was achieved in two TNBC cell lines, and although cell viability was unaffected, defects in receptor-mediated endocytosis were observed. EGFR signaling to Erk and Akt kinases was impaired in Endo II KD cells, and this correlated with reduced rates of EGFR internalization and cell motility. Endo II KD cells also displayed defects in three dimensional (3D) cell invasion, and this correlated with impaired extracellular matrix degradation and internalization of MT1-MMP. Endo II silencing also caused a significant reduction in TNBC tumor growth and lung metastasis in mammary orthotopic tumor xenograft assays. In human breast tumor specimens, Endo II expression was highest in TNBC tumors compared with other subtypes, and at the level of gene expression, high Endo II was associated with reduced relapse-free survival in patients with basal-like breast cancers. Together, these results identify a positive role for Endo II in TNBC tumor metastasis and a potential link with poor prognosis. Endophilin A2 and related adaptor proteins represent important signaling hubs to target in metastatic cancers.

Disabled-2 negatively regulates lipopolysaccharide-stimulated Toll-like receptor 4 signaling and inflammation response in murine macrophage (INM7P.349)

Abstract Toll-like receptor 4 (TLR4)-mediated host response to lipopolysaccharide (LPS) stimulation plays a pivotal role in host defense against from Gram-negative bacterial infection. Upon ligand binding, TLR4/MD2 complexes are internalized to form endocytic signalsomes and extend LPS response. However, the underlying mechanism of LPS-stimulated TLR4 internalization and signaling has not yet been completely understood. Here we delineated whether the endocytic adaptor protein Disabled-2 (Dab2) that is highly expressed in macrophage plays a role in TLR4 signaling and trafficking. In vivo analysis of the myeloid lineage-restricted Dab2 knockout (Dab2-/-) mice revealed that the mice were more susceptible to LPS challenge leading to an overproduction of the chemokine RANTES in serum and poor survival. The functional role of Dab2 in TLR4 signaling was investigated further using RAW264.7 macrophage with Dab2 knockdown. We found that attenuation of Dab2 expression increased the basal level of cell surface TLR4/MD2 complexes and accelerated the rate of LPS-induced down-regulation of these complexes from the cell surface. Consequently, TLR4 endocytic pathway-dependent IRF-3 activation was augmented with an increased expression for the subsets of inflammatory cytokine and IFN-inducible genes. These data indicate that Dab2 functions as a negative regulator of TLR4 endocytic pathway and signaling, supporting a novel role of Dab2 in the inflammatory response of macrophage to endotoxin.

Familial Hyperkalemic Hypertension (FHHt) Proteins Form an Endocytic Switch for the Renal Excretory Channel, KCNJ1

The kidney potassium channel, ROMK (KCNJ1), controls urinary potassium excretion, in states of dietary potassium deficiency the channel is down‐regulated by the endocytic clathrin adaptor protein, ARH. Inappropriate ROMK endocytosis contributes to hyperkalemia in Familial Hyperkalemic Hypertension (FHHt), a rare disorder caused by mutations in WNK 1, CUL3, and KLHL3. Here we explore how WNK1 and the E3 ligase complex containing CUL3 / KLHL3 regulate ARH ubiquitination, abundance, and ROMK expression. We found ARH directly binds to and is ubiquitinated by the CUL3/ KLHL3 E3 ligase complex, leading to ARH degradation. WNK1 blocks ARH degradation and KLHL3 binding by controlling phosphorylation of ARH at serine 14. Phosphorylation of ARH led to increased abundance as did the S14D phosphomimetic by blocking the direct ARH‐KLHL3 interaction. Further we found WNK1 activates a kinase cascade resulting in the direct phosphoryation of ARH S‐14 by the ERK5 kinase, as demonstrated by in vitro phosphorylation assays with recombinant ERK5 and in cells with the ERK5 inhibitor BIX02189, a finding consistent with the observed increase in activated ERK5 and ARH in mice fed a low K+ diet. These studies provide a molecular insight to understand how mutations in WNK1 and CUL3‐KLHL3 acerbate channel endocytosis and disrupt potassium homeostasis in FHHt.

Intersectin adaptor proteins are associated with actin-regulating protein WIP in invadopodia

Invasive cancer cells form actin-rich membrane protrusions called invadopodia that degrade extracellular matrix and facilitate cell invasion and metastasis. WIP (WASP-interacting protein) together with N-WASP (neural Wiskott-Aldrich syndrome protein) are localized in invadopodia and play a crucial role in their formation. Here we show that WIP interacts with endocytic adaptor proteins of the intersectin (ITSN) family, ITSN1 and ITSN2. The interaction is mediated by the SH3 domains of ITSNs and the middle part of the WIP proline-rich motifs. We have also demonstrated that ITSN1, WIP and N-WASP can form a complex in cells. Endogenous ITSN1 and ITSN2 are located in invasive protrusions of MDA-MB-231 breast cancer cell line. Moreover, data from immunofluorescent analysis revealed co-localization of ITSN1 and WIP at sites of invadopodia formation and in clathrin-coated pits. Together, these findings provide insights into the molecular mechanisms of invadopodia formation and identify ITSNs as scaffold proteins involved in this process.

Comprehensive identification of phosphorylation sites on the Numb endocytic adaptor protein.

Numb is an adaptor protein that functions in the endocytosis and intracellular trafficking of membrane receptors and adhesion molecules. Previous studies have indicated that Numb localization and function are regulated through phosphorylation by atypical protein kinase C at several key sites. Here, using LC-MS/MS, we report the identification of 25 serine/threonine Numb phosphorylation sites, and a single tyrosine phosphorylation site. Amino acid sequences flanking several of the sites identified conform to consensus motifs for cyclin-dependent kinase 5 (CDK5). In vitro kinase assays and immunoblotting confirmed that CDK5 phosphorylates Numb. LC-MS/MS analysis identified specific CDK5-directed phosphorylation of Numb at position S288 and at two additional regions. Therefore, Numb is likely to exist in multiple phospho-isoforms, and may be subject to phosphorylation-mediated regulation downstream of CDK5. These findings provide a basis for further investigations into the complex role of Numb phosphorylation in regulating its subcellular localization, protein interactions, and function. All MS data have been deposited in the ProteomeXchange with identifier PXD000997 (http://proteomecentral.proteomexchange.org/dataset/PXD000997).

Numb regulates the polarized delivery of cyclic nucleotide-gated ion channels in rod photoreceptor cilia.

The development and maintenance of protein compartmentalization is essential for neuronal function. A striking example is observed in light-sensing photoreceptors, in which the apical sensory cilium is subdivided into an inner and outer segment, each containing specific proteins essential for vision. It remains unclear, however, how such polarized protein localization is regulated. We report here that the endocytic adaptor protein Numb localizes to the inner, but not the outer segment of mouse photoreceptor cilia. Rod photoreceptor-specific inactivation of numb in vivo leads to progressive photoreceptor degeneration, indicating an essential role for Numb in photoreceptor cell biology. Interestingly, we report that loss of Numb in photoreceptors does not affect the localization of outer segment disk membrane proteins, such as rhodopsin, Peripherin-rds, Rom-1, and Abca4, but significantly disrupts the localization of the rod cyclic nucleotide-gated (Cng) channels, which accumulates on the inner segment plasma membrane in addition to its normal localization to the outer segments. Mechanistically, we show that Numb interacts with both subunits of the Cng channel and promotes the trafficking of Cnga1 to the recycling endosome. These results suggest a model in which Numb prevents targeting of Cng channels to the inner segment, by promoting their trafficking through the recycling endosome, where they can be sorted for specific delivery to the outer segment. This study uncovers a novel mechanism regulating polarized protein delivery in light-sensing cilia, raising the possibility that Numb plays a part in the regulation of protein trafficking in other types of cilia.

UIM domain-dependent recruitment of the endocytic adaptor protein Eps15 to ubiquitin-enriched endosomes.

BackgroundEps15 is an endocytic adaptor protein that stimulates clathrin-mediated endocytosis. Among other interactions, Eps15 binds ubiquitin via UIM domains, recruiting ubiquitinated cargo into clathrin-coated vesicles. In EGF-treated cells, Eps15 also localizes to endosomes. The basis of this localization is not known.ResultsWe show that accumulation of ubiquitinated cargo can recruit Eps15 to endosomes via UIM domain interactions. First, treatment of SK-Br-3 breast cancer cells, which overexpress the EGFR family member ErbB2, with geldanamycin to promote receptor ubiquitination and endosomal transport, recruited FLAG-Eps15 to endosomes. Two in-frame ubiquitin constructs, PM-GFP-Ub (retained in endosomes after endocytosis), and GFP-FYVE-UbΔGG (targeted directly to endosomes) also recruited Eps15 to endosomes, as did slowing endosome maturation with constitutively-active Rab5-Q79L. Endosomal recruitment required the UIM domains, but not the N-terminal EH domains or central coiled-coil domains, of Eps15. Silencing of the endosomal Eps15 binding partner Hrs did not affect recruitment of Eps15 to ubiquitin-enriched endosomes. In fact, Hrs silencing itself modestly recruited Eps15 to endosomes, probably by accumulating endogenous ubiquitinated cargo. Eps15 silencing did not affect lysosomal degradation of ubiquitinated ErbB2; however, GFP-FYVE-UbΔGG overexpression inhibited internalization of EGFR and transferrin receptor.ConclusionsWe show for the first time that ubiquitin is sufficient for Eps15 recruitment to endosomes. We speculate that Eps15 recruitment to ubiquitin-rich endosomes may reduce the level of Eps15 at the plasma membrane, slowing endocytosis to allow time for processing of ubiquitinated cargo in endosomes.