Browsing by Subject "Endocytosis"
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Item Characterization of Alsin and Its Role in IGF-1-Mediated Neuronal Survival(2005-04-29) Topp, Justin David; Horazdovsky, BruceThe transport of proteins between organelles is a highly regulated and complex process that is crucial for many of the functions required for cellular homeostasis. Many distinct proteins are involved in each trafficking step with roles in vesicle formation, budding, movement, and fusion. One class of proteins, the Rab GTPases, is required for docking and fusion of transport vesicles with their target membrane. These proteins are regulated by their state of nucleotide binding, with GTP-bound Rabs thought to provide specificity to transport steps via their interactions with specific effector proteins. While much work has been focused on proteins downstream of Rab GTPases, little is known as to how the activation of these proteins is controlled. This is particularly true of Rab5, the Rab protein required for vesicle fusion at the endosome. Endocytosis of plasma membrane proteins requires Rab5(GTP, and humans possess at least seven proteins (Vps9 family) that are expected to activate Rab5. An intriguing aspect of the Vps9 family of proteins is that they appear to link signal transduction to receptor trafficking via the specific coupling of particular receptors to Rab5-mediated endocytosis. Cell biological, biochemical, and immunohistochemical techniques were employed to characterize one of the Vps9 family proteins named Alsin. Alsin is required for motor neuron maintenance and/or survival, as loss-of-Alsin function results in multiple juvenile-onset neurodegenerative disorders (ALS2, JPLS, IAHSP). It was found here that Alsin is an endosomal protein that activates both Rac1 and Rab5. This protein is present in all of the tissues associated with the aforementioned diseases and intriguingly is upregulated in the cerebellum, an unknown site of pathology for this class of disorders. Alsin was found to couple Rab5 activation specifically to the IGF-1 signal transduction pathway via its regulation of IGF-1 receptor endocytosis. This function of Alsin was shown to be essential for IGF-1-mediated cell survival. These results provide the first characterization of Alsin and identify a novel cause for neurodegeneration.Item Genetic Dissection of Synaptic Vesicle Endocytosis(2019-03-28) Afuwape, Olusoji Adeyemi; Terman, Jonathan R.; Kavalali, Ege T.; Schmid, Sandra; Monteggia, Lisa; Krämer, HelmutSynaptic transmission is mediated by the quantal release of neurotransmitters through the fusion of discrete synaptic vesicles with the presynaptic membrane. To maintain reliable transmission, synaptic vesicles and proteins must be recycled after release of neurotransmitters. A key protein in this recycling process is dynamin. Dynamin is a GTPase that catalyzes the scission of a budding endosome off its parent membrane. The mammalian brain expresses three isoforms of dynamin. Using genetically modified mouse hippocampal neurons, I analyzed the functional significance of dynamin in synaptic vesicle endocytosis. Specifically, I assessed dynamin 2 function in synaptic vesicle recycling and neurotransmission and investigated the role of dynamin independent endocytosis at the synapse. My data demonstrates that synaptic transmission after post-natal knockout of dynamin 2 remains intact and synaptic vesicle endocytosis, assessed by the trafficking of vesicular glutamate transporter fused to pHluorin, is unperturbed. Synaptic vesicle endocytosis in the absence of dynamin 2 was assessed at both room temperature and 32 oC. At both temperatures, my results reveal that synaptic vesicle recycling functions independent of dynamin 2 but also, the kinetics of single vesicle recycling is unaffected by changes in temperature suggesting that a single, temperature insensitive (within the limits of testing) form of endocytosis mediates single synaptic vesicle endocytosis. Further experiments reveal that the retrieval of single synaptic vesicles persists after the knockout of all dynamin isoforms. However, after multivesicular release, my results show an overall decrease in synaptic vesicle pool size and a retardation of subsequent vesicle endocytosis in neurons lacking all dynamin isoforms suggesting dynamin function at the synapse is activity dependent. This finding is consistent with prior reports showing dynamin function at the synapse is dependent on its dephosphorylation by the Ca2= dependent phosphatase, calcineurin. My results also demonstrate a dichotomy in the dependence of dynamin for synaptic neurotransmission. Whereas I observe a decrease in evoked amplitude, release probability and frequency of spontaneously released events in glutamatergic synapses, I observe no discernable defects in GABAergic neurotransmission. This result suggests inhibitory synapses are better equipped with compensatory mechanisms to deal with the loss of dynamins 1,2 and 3. Overall, my data demonstrate that dynamin is crucial but not essential for synaptic vesicle endocytosis. Dynamin is an activity dependent GTPase that is required for synaptic vesicle recycling after exocytosis of multiple vesicles. However, the underlying mechanism of single synaptic vesicle endocytosis is both dynamin and temperature independent.Item How to Mend a Broken Heart: Massive Endocytosis and the Role of Lipidic Forces in Membrane Trafficking(2012-07-20) Fine, Michael Jon; Hilgemann, Donald W.Novel forms of membrane internalization defined as massive endocytosis (MEND) were characterized for mechanism and physiological significance in isolated cells and intact cardiac tissue. These non-canonical forms of membrane trafficking are related to perturbations within the outer lipid monolayer of the plasmalemma or through coupled calcium-mediated fusion and phosphatidylinositol 4,5-bisphosphate (PIP2) signaling. Both forms of MEND do not rely on classical endocytic proteins such as clathrin or dynamin and appear independent of the cytoskeletal or extracellular matrix. A hypothesis emerged implicating lipidic driven processes as the mechanistic basis of MEND. One explanation involves lipid domain formation with excessive inward curvature from rapid accumulation of PIP2 within the inner monolayer subsequent to large calcium transients. Alternatively, perturbation of the outer monolayer through amphipathic detergents or modulation of lipid content also promotes inward curvature of the membrane leading to massive endocytosis. Electrophysiological and optical methods support MEND preferentially internalizing membrane of liquid-ordered (Lo) domains leading to the potential selection of certain proteins and markers due to physiological segregation into their respective energetically favorable domains. MEND demonstrates that lipidic reorganization of the membrane may be sufficient for rapid and selective internalization of the plasma membrane. The implications of such a novel form of endocytosis could dramatically impact numerous physiological and cellular activities. In cardiac pathologies, short-term changes in the surface membrane expression of vital ionic transporters have been implicated in hypertrophy, atrial defibrillation, and damage post myocardial ischemia. It is possible that the properties underlying MEND mechanics may be responsible for some of these acute cardiac changes. Protocols that induce massive endocytosis were performed on isolated cardiac myocytes with similar results described in the fibroblast cell lines. MEND occurs in cardiomyocytes with calcium transients. MEND also occurs upon isolation of myocytes without stimulation. While this indicates that MEND does occur in primary cells, protocols to mimic MEND in intact cardiac tissue have remained inconclusive. Cellular processes during the isolation procedures cause dramatic changes in the membrane of cardiac myocytes. Subsequently, it is likely isolated cardiomyocytes have distinct differences from unstressed intact tissue. MEND may still occur in cardiac tissue as well as other physiological systems and newer protocols to monitor membrane movements in intact tissues are currently being investigated.Item Illuminating Endocytic Organelles with pH-Resposive [sic] Nanomaterials(2017-02-20) Wang, Chensu; DeBerardinis, Ralph J.; White, Michael A.; Gao, Jinming; Danuser, Gaudenz; Yoo, Hyuntae; Zhong, QingEndosomes, lysosomes and related catabolic organelles are a dynamic continuum of vacuolar structures that impact a number of key cell physiological processes that include protein/lipid metabolism, nutrient sensing and cell survival. To support quantitative investigation of these processes in living cells, we have developed a library of ultra-pH sensitive (UPS) fluorescent nanoparticles with chemical properties that allow fine-scale, multiplexed, spatial-temporal perturbation and quantification of catabolic organelle maturation at single organelle resolution. Deployment in cells enabled quantification of the proton accumulation rate in endosomes; illumination of previously unrecognized regulatory mechanisms coupling pH transitions to endosomal coat protein exchange; discovery of distinct pH thresholds required for mTORC1 activation by free amino acids versus proteins; broad-scale characterization of the consequence of endosomal pH transitions on cellular metabolomic profiles; and functionalization of a context-specific metabolic vulnerability in lung cancer cells. These biological applications benchmarked the robustness and adaptability of this nanotechnology-enabled 'detect and perturb' strategy. As a translational application, we leveraged the technology in high-throughput screening assays that successfully identified chemical agents in the promotion of autophagolysosomal activity through TFEB activation. Formulation of these compounds in liver-tropic biodegradable, biocompatible nanoparticles conferred hepatoprotection against diet-induced steatosis in murine models and prolonged survival in Caenorhabditis elegans. These results highlight the therapeutic potential of small-molecule TFEB activators to ameliorate metabolic syndrome and extend lifespan.Item Methods for Identifying Subcellular Targeting Ligands and Selected Applications(2015-09-09) Umlauf, Benjamin J.; Albanesi, Joseph P.; Brown, Kathlynn C.; Schmid, Sandra; Kohler, Jennifer J.Subcellular localization plays an essential role in targeting drug therapies as generally the pro-drug or linker relies on physical conditions of a particular subcellular compartment to function. We developed two methods that allow for selecting targeting ligands that both internalize specifically in cancer cells as well as accumulate in a defined subcellular location. The first method utilizes endocytic selection pressure to identify targeting peptides from a phage displayed peptide library that internalize specifically in cancer cells via a defined mechanism of endocytosis while the second couples together a traditional biopanning selection protocol with a secondary immunofluorescent screen to directly identify functional targeting peptides. In addition we also present a method for reducing amplification bias during phage display experiments. Finally, we demonstrate the utility of a novel peptide, termed H1299.3 selected via the endocytic pressure method, to serve as targeting ligand for a novel immunotherapy. H1299.3 targeted liposomes facilitate delivery of antigenic peptide specifically in MHC class 1 molecules of cancer cells resulting in activation of secondary immune response against the cancer cells. Thus, we present two novel methods for identify targeting ligands with selective internalization that accumulate in defined subcellular localizations as well as a novel application for a newly identified targeting ligand.Item Molecular Determinants of Synaptic Vesicle Exocytosis and Endocytosis Coupling(2016-07-12) Li, Ying Chuan; Rizo-Rey, José; Krämer, Helmut; Xu, WeiSynaptic vesicle recycling is essential for maintaining normal synaptic function. The reuse of vesicles maintains the size of the presynaptic terminal and ensures the availability of synaptic vesicles for subsequent exocytosis. The coupling of exocytosis and endocytosis allows for continued rapid synaptic transmission; however, the molecular mechanisms of this process are not well understood. This coupling is assumed to be Ca2+-dependent but the exact role of Ca2+ and its key effectors in the regulation of endocytosis are not clear. Using a genetically encoded pH-sensitive GFP tag expressed in cultured hippocampal neurons, I analyzed synaptic vesicle trafficking in high resolution optical experiments. By manipulating the expression of various effectors of vesicle fusion I was able to dissect out the relationship between exocytic pathway and subsequent endocytic kinetics. My results showed that the slowed endocytosis phenotype previously reported after synaptotagmin 1 loss-of-function can also be triggered by other manipulations that promote asynchronous release such as Sr2+ substitution and complexin loss-of-function. The link between asynchronous release and slowed endocytosis was due to selective targeting of fused synaptic vesicles towards slow retrieval by the asynchronous release Ca2+ sensor synaptotagmin7. This divergence in Ca2+ sensor function supports findings that VAMP4 selectively drive asynchronous release through a population of vesicles that do not interact with synaptotagmin 1 or complexins. At the single synaptic vesicle level, synaptotagmin 1 acted as an essential determinant of synaptic vesicle endocytosis time course by increasing the kinetics of vesicle retrieval in response to increasing Ca2+ levels. In contrast, synaptotagmin 1 did not affect the rapid retrieval of spontaneously fused vesicles. Taken together, these results suggest that exocytic pathways dictate endocytic kinetics as asynchronously fused vesicles are retrieved slowly while spontaneously fused vesicles are rapidly retrieved. These mechanisms may diversify the molecular compositions of synaptic vesicles regenerated after fusion to provide presynaptic terminals with a wide range of synaptic vesicle populations with distinct biogenesis properties and exo-endocytosis kinetics.Item Optical Quantal Analysis of Evoked and Spontaneous Single-Vesicle Fusion(2014-07-14) Leitz, Jeremy Thomas Sheng; Hilgemann, Donald W.; Hiesinger, Peter Robin; Albanesi, Joseph P.; Kavalali, Ege T.Synaptic vesicle recycling is critical for the maintenance and proper function of neurotransmission. Neurotransmission can proceed through action-potential evoked vesicle fusion where, upon depolarization, Ca2+ enters the nerve terminal though voltage-gated channels, interacts with vesicle-associated proteins to promote fusion with the terminal membrane, and causes release of vesicle contents. Neurotransmission can also occur spontaneously in the absence of stimulation, although this process is still Ca2+-dependent. Regardless of the mode of vesicle fusion, the vesicle lipids and protein components must be removed from the terminal membrane; the vesicle must be reconstituted and re-filled with neurotransmitter, so that it may ultimately be reused. Uncoupling the roles of Ca2+ in synaptic vesicle fusion and retrieval has been difficult to date as studies have relied on measurements of bulk synaptic vesicle retrieval. Here, to dissect the role of Ca2+ in these processes, we utilized low signal-to-noise pHluorin-tagged vesicular probes to monitor single synaptic vesicle recycling of both action-potential evoked and spontaneous fusion vesicles in rat hippocampal neurons. We show that during stimulation, increasing extracellular Ca2+ increases synaptic vesicle fusion probability, but decreases the rate of synaptic vesicle retrieval. This negative regulation of synaptic vesicle retrieval is blocked by the Ca2+ chelation as well as inhibition of calcineurin, a Ca2+-calmodulin-dependent phosphatase. Indeed, the slow time course of aggregate synaptic vesicle retrieval detected during repetitive activity can be explained by a progressive decrease in the rate of synaptic vesicle retrieval during the stimulation train. These results indicate Ca2+ entry during single action potentials slows the pace of subsequent synaptic vesicle recycling. Conversely, we found that synaptic vesicles that undergo spontaneous fusion are retrieved very rapidly and this retrieval time is Ca2+-independent. Interestingly, we found that within a single synaptic bouton, the rate of spontaneous neurotransmission is independent of evoked fusion probability, suggesting there are fundamental regulatory differences between these forms of neurotransmission. Moreover, we found that the glycoprotein Reelin can act presynaptically to enhance spontaneous neurotransmission without affecting evoked neurotransmission by mobilizing a molecularly specific subset of synaptic vesicles. These data illustrate fundamental differences in vesicle recycling between modes of neurotransmission at the single-vesicle level.Item Quantitative Analysis of Integrin Trafficking and Focal Adhesion Turnover to Study Integration of Early Endocytic Trafficking, Signaling and Migration(2019-01-22) Lakoduk, Ashley Marie; Danuser, Gaudenz; Ross, Theodora; Rosen, Michael K.; Schmid, SandraEarly endocytic trafficking is critical for regulating both receptor presence at the plasma membrane and cellular signaling. Clathrin-mediated endocytosis (CME) constitutes a major route of selective receptor internalization, yet little is knowing about how this process is regulated both temporally and spatially. We chose to investigate focal adhesion (FA) and beta-1 integrin turnover as a model system to better understand the spatial, temporal, and cargo-specific regulation of early endocytic trafficking. Importantly, the dynamic turnover of integrins and their associated adhesion complexes through endocytic and recycling pathways has emerged as key mechanism for controlling cancer cell migration and invasion. However, current tools available to study integrin trafficking do not provide adequate spatial information or can induce artifacts. Here, we report the generation and characterization of a neutral and monovalent antibody-based probe to study beta-1 integrin trafficking in cells. Using this tool, we report a novel regulatory mechanism of integrin turnover by a lipid kinase, PIPKI, and discover a mutant p53-driven endosomal signaling nexus that regulates beta-1 integrin recycling and cancer cell invasion. This work demonstrates the importance of the reciprocal crosstalk between early endocytic trafficking and receptor signaling in regulating both normal and tumor cell function.Item Rab5 Activation by the Vps9 Domain(2007-05-21) Carney, Darren Scott; Horazdovsky, BruceThe movement of proteins through the endocytic pathway is a complex and highly regulated process. Not only is this pathway used to internalize cellular nutrients, but it is also used to modulate a cell's response to extracellular stimuli. Internalization and subsequent trafficking of transmembrane receptor proteins that receive these signals from the external milieu play an essential role in establishing and maintaining cellular homeostasis. As key regulators of the early stages the endocytic pathway, the small GTPases of the Rab5 family serve an essential function in integrating intracellular protein traffic and these cell signaling events. Rab5 proteins exert their influence on protein trafficking only when bound to GTP. A large family of proteins containing a conserved domain (Vps9) activate Rab5 by promoting the release of GDP and reloading of GTP. These nucleotide exchange factors contain additional domains which link them to specific cellular locations or signaling cascades. The multiplicity of these Rab5 proteins and exchange factors raises the question of how these proteins specifically interact to regulate individual trafficking events. To investigate this specificity, the three Rab5 proteins of Saccharomyces cerevisiae, Vps21, Ypt52 and Ypt53, and the two yeast Vps9 domain-containing proteins, Vps9 and Muk1, were analyzed. This analysis identified previously unappreciated roles for Ypt53 and Muk1 in a relatively late stage of endocytosis. A mutational analysis of Vps9 identified several residues important for Vps9 domain function and shed light on a possible intramolecular regulation of this domain by the carboxy-terminal ubiquitin-binding CUE domain. Finally, structural studies of the Rab5/Vps9 domain complex were initiated to gain a better understanding of the molecular mechanisms by which Rab5 proteins interact with and are activated by the Vps9 domain.Item Regulation of Clathrin Mediated Endocytosis and Its Role in Cancer Progression(2017-05-15) Connelly, Sarah Elkin; Minna, John D.; Schmid, Sandra; Shay, Jerry W.; Danuser, GaudenzMetastasis is a multistep process requiring cancer cell signaling, invasion, migration, survival, and proliferation. These processes require dynamic modulation of cell surface proteins by endocytosis. Given this functional connection, it has been suggested that endocytosis is dysregulated in cancer. To test this, we developed In-Cell ELISA assays to measure three different endocytic pathways: clathrin-mediated endocytosis, caveolae-mediated endocytosis, and clathrin-independent endocytosis and compared these activities in 29 independently isolated non-small cell lung cancer (NSCLC) cell lines to determine whether there were systematic changes in the three different endocytic pathways. However we observed significant heterogeneity. Nonetheless, using hierarchical clustering based on their combined endocytic properties we identified two phenotypically distinct clusters of NSCLCs. One co-clustered with mutations in KRAS, a mesenchymal phenotype, increased invasion through collagen and decreased growth in soft agar, whereas the second was enriched in cells with an epithelial phenotype. We also used the In-Cell ELISA assay to characterize Ikarugamycin (IKA), a previously discovered antibiotic, which inhibits the uptake of oxidized low-density lipoproteins in macrophages, as well as clathrin-mediated endocytosis (CME) in plant cell lines. However, detailed characterization of IKA had yet been performed. Therefore, we performed biochemistry and microscopy experiments to further characterize the effects of IKA on CME. We showed that IKA acutely inhibits CME, but not other endocytic pathways with an IC50 of 2.7 μM. Although long-term incubation with IKA has cytotoxic effects, the short-term inhibitory effects on CME were reversible. Thus, IKA can be a useful tool for probing routes of endocytic trafficking. Finally, we investigated possible mechanisms that lead to altered endocytosis in cancer cells. We discovered that dynamin 1 (Dyn1), previously thought to be neuron specific is frequently upregulated and postranslationally regulated in cancer cells. Dyn1 expression alters the proliferation rates, growth in soft agar, and tumor growth of cancer cells. We hypothesize that these changes are due to alteration in cell surface protein expression and downstream signaling pathways and have developed protocols to test these hypothesizes. Taken together, our results suggest that endocytic alterations in cancer cells can significantly influence cancer-relevant phenotypes.Item Regulation of Endocytic Recycling by FGD4, a Cdc42 GEF(2012-07-10) Cardenas, Jessica A.; White, Michael A.The family of Receptor Tyrosine Kinases (RTKs) are a group of cell surface receptors with the capability of activating, through phosphorylation, multiple kinase cascades in response to activation by an extracellular ligand. This allows a cell to respond to its environment and induce a range of cellular processes such as proliferation, differentiation, migration, and apoptosis. Unsurprisingly, these powerful transducers of extracellular signaling are often found mutated in human disease, such as cancer. Therefore, learning how these receptors are downregulated and processed once they have been activated may provide novel avenues of therapeutic intervention. How receptors are processed after internalization and fusion into the sorting endosome (also known as the early endosome) still largely remains unknown. Here, we discovered a Cdc42 GEF, FGD4, that may be important for shuttling ErbB receptors to the recycling endosome via a microtubule dependent mechanism. Through protein depletion studies we show that FGD4 is important for mitosis, microtubule stability, migration and endocytic trafficking of EGF, an ErbB1 ligand. Dynamic microtubule regulation are critical in these cell biological process, therefore we hypothesize that FGD4 may be regulating these diverse cell functions via a microtubule dependent mechanism.Item Regulation of Erythropoietin Receptor Endocytosis and Signaling(2009-01-14) Sulahian, Rita; Huang, LilyErythropoietin (Epo) and its receptor (EpoR) play an essential role in the survival, proliferation and differentiation of precursor red blood cells into mature erythrocytes. EpoR lacks intrinsic enzymatic activity essential to mediate downstream signaling cascades, instead, it associates with Janus tyrosine kinase 2 (JAK2), which upon Epo binding is auto-phosphorylated and activated. Activated JAK2 phosphorylates many of the tyrosines in the EpoR which recruit SH2-domain containing proteins that will carry on the signaling event. EpoR -/- and JAK2 -/- mice die during embryogenesis due to the absence of definitive erythropoiesis. Once activated, the EpoR is internalized and degraded through unidentified mechanisms. Sustained EpoR signaling and aberrant JAK2 activation are observed in hematological malignancies. Here we show that cell-surface EpoR is internalized via clathrin-mediated endocytosis. Ligand-dependent EpoR internalization requires both JAK2 kinase activity and EpoR cytoplasmic tyrosines. In addition, phosphorylated Y429, Y431, and Y479 in the EpoR cytoplasmic domain binds p85 subunit of phosphoinositide 3-kinase (PI3-kinase) upon Epo stimulation and individually is sufficient to mediate Epo-dependent EpoR internalization. Knockdown of p85 alpha and p85 beta but not inhibition of PI3-kinase activity dramatically impaired EpoR internalization, indicating that p85 alpha and p85 beta may recruit proteins in the endocytic machinery upon Epo stimulation. We carried on a candidate siRNA screen for endocytic proteins involved in EpoR endocytosis. c-Cbl, a E3 ubiquitin ligase associated with p85, was identified to be essential in mediating EpoR internalization. Ligase-deficient c-Cbl mutants dramatically diminished ligand-induced EpoR internalization. Consistent with this finding, c-Cbl knockout animals exhibit erythroid hyperplasia. We further demonstrate that ubiquitination of the EpoR itself is not required for internalization but may be important for endosomal sorting and degradation, and K428 in the EpoR cytoplasmic domain may be the primary ubiquitination site. Further studies will uncover the role of c-Cbl in EpoR down-regulation. These results provide the first characterization of EpoR downregulation mechanisms.Item Ubiquitination of EpoR and p85 in Ligand Induced EpoR Down-Regulation(2014-07-09) Bulut, Gamze Betul; Yin, Helen L.; Huang, Lily; Zhang, Chengcheng "Alec"; Seemann, JoachimErythropoietin (Epo) is the primary cytokine that drives red blood cell production and signals through its receptor, the EpoR, on erythroid progenitor cells. Epo binding to EpoR activates Janus kinase 2 (JAK2), which phosphorylates cytoplasmic tyrosines of the EpoR. Signaling proteins bind these phosphotyrosines through SH2 domains, leading to the survival and proliferation of erythroid progenitor cells and the differentiation of these progenitors into mature erythrocytes. Therefore, EpoR signaling is essential for red blood cell production. To maintain physiologic numbers of circulating red blood cells EpoR signaling is also subject to negative regulation. Mutations in EpoR or JAK2 that abrogate negative regulation cause erythrocytosis in hematological malignancies. Primary familial and congenital polycythemia (PFCP) is a proliferative disorder characterized by erythrocytosis and hypersensitivity of erythroid progenitors to Epo. Defects in negative regulation of EpoR signaling contribute to the etiology of PFCP. However, the underlying molecular mechanisms are poorly understood. Here we show that ubiquitination of EpoR controls internalization, lysosomal sorting, degradation and signaling of the EpoR. Ubiquitination of EpoR at K256 is necessary and sufficient for efficient Epo-induced receptor internalization, while ubiquitination at K428 promotes trafficking of activated receptors to the lysosomes for degradation. Interestingly, EpoR that cannot be ubiquitinated has reduced mitogenic activities and ability to stimulate the downstream signaling pathways. We propose that ubiquitination of the EpoR critically controls both receptor down-regulation and signaling. Secondly, we identified a novel mechanism mediating Epo-dependent EpoR internalization. Epo induces Cbl-dependent ubiquitination of the p85, which binds to phosphotyrosines on EpoR. Ubiquitination allows p85 to interact with epsin-1, thereby driving EpoR endocytosis. Knockdown of Cbl, expression of its dominant negative forms, or expression of an epsin-1 mutant all compromise Epo-induced EpoR internalization. Mutated EpoRs mimicking those from PFCP patients cannot bind p85, co-localize with epsin-1, nor internalize upon Epo stimulation and exhibit Epo hypersensitivity. Restoring p85 binding to PFCP receptors rescues Epo-induced epsin-1 co-localization, EpoR internalization, and normalizes Epo hypersensitivity. Our results uncover the role of EpoR ubiquitination and a novel Cbl/p85/epsin-1 pathway in EpoR endocytosis and show that defects in this pathway contribute to excessive Epo signaling and erythroid hyperproliferation in PFCP.