The Cbl protooncoprotein stimulates CSF-1 receptor multiubiquitination and endocytosis, and attenuates macrophage proliferation.

Colony-stimulating factor-1 (CSF-1) activation of the CSF-1 receptor (CSF-1R) causes Cbl protooncoprotein tyrosine phosphorylation, Cbl-CSF-1R association and their simultaneous multiubiquitination at the plasma membrane. The CSF-1R is then rapidly internalized and degraded, whereas Cbl is deubiquitinated in the cytoplasm without being degraded. We have used primary macrophages from gene-targeted mice to study the role of Cbl. Cbl-/- macrophages form denser colonies and, at limiting CSF-1 concentrations, proliferate faster than Cbl+/+ macrophages. Their CSF-1Rs fail to exhibit multiubiquitination and a second wave of tyrosine phosphorylation previously suggested to be involved in preparation of the CSF-1-CSF-1R complex for endocytosis. Consistent with this result, Cbl-/- macrophage cell surface CSF-1-CSF-1R complexes are internalized more slowly, yet are still lysosomally degraded, and the CSF-1 utilization by Cbl-/- macrophages is reduced approximately 2-fold. Thus, attenuation of proliferation by Cbl is associated with its positive regulation of the coordinated multiubiquitination and endocytosis of the activated CSF-1R, and a reduction in the time that the CSF-1R signals from the cell surface. The results provide a paradigm for studies of the mechanisms underlying Cbl attenuation of proliferative responses induced by ligation of receptor tyrosine kinases.     

Dual Roles for RHOA/RHO-Kinase In the Regulated Trafficking of a Voltage-sensitive Potassium Channel

Kv1.2 is a member of the Shaker family of voltage-sensitive potassium channels and contributes to regulation of membrane excitability. The electrophysiological activity of Kv1.2 undergoes tyrosine kinase-dependent suppression in a process involving RhoA. We report that RhoA elicits suppression of Kv1.2 ionic current by modulating channel endocytosis. This occurs through two distinct pathways, one clathrin-dependent and the other cholesterol-dependent. Activation of Rho kinase (ROCK) via the lysophosphatidic acid (LPA) receptor elicits clathrin-dependent Kv1.2 endocytosis and consequent attenuation of its ionic current. LPA-induced channel endocytosis is blocked by the ROCK inhibitor Y27632 or by clathrin RNA interference. In contrast, steady-state endocytosis of Kv1.2 in unstimulated cells is cholesterol dependent. Inhibition of basal ROCK signaling with Y27632 increased surface Kv1.2, an effect that persists in the presence of clathrin small interfering RNA and that is not additive to the increase in surface channel levels elicited by the cholesterol sequestering drug filipin. Temperature block experiments show that ROCK affects cholesterol-dependent trafficking by modulating the recycling of endocytosed channel back to the plasma membrane. Both receptor-stimulated and steady-state Kv1.2 trafficking modulated by RhoA/ROCK required the activation of dynamin as well as the ROCK effector Lim-kinase, indicating a key role for actin remodeling in RhoA-dependent Kv1.2 regulation.     

Abl tyrosine kinase regulates endocytosis of the epidermal growth factor receptor

Signal attenuation from ligand-activated epidermal growth factor receptor (EGFR) is mediated in part by receptor endocytosis and trafficking to the lysosomal degradative compartment. Uncoupling the activated EGFR from endocytosis and degradation has emerged as a mechanism for oncogenic activation of the EGFR. The Abl nonreceptor tyrosine kinase is activated by ligand-stimulated EGFR, but the role of Abl in EGFR signaling has not been defined. Here we uncovered a novel role for the activated Abl kinase in the regulation of EGFR endocytosis. We show that activated Abl impairs EGFR internalization. Moreover, we show that activated Abl phosphorylates the EGFR primarily on tyrosine 1173, and that mutation of this site to phenylalanine restores ligand-dependent endocytosis of the EGFR in the presence of activated Abl. Furthermore, we show that activated Abl allows the ligand-activated EGFR to escape Cbl-dependent down-regulation by inhibiting the accumulation of Cbl at the plasma membrane in response to epidermal growth factor stimulation and disrupting the formation of the EGFR.Cbl complex without affecting Cbl protein stability. These findings reveal a novel role for Abl in promoting increased cell-surface expression of the EGFR and suggest that Abl/EGFR signaling may cooperate in human tumors.     

Endocytosis and cancer: an 'insider' network with dangerous liaisons

From the signaling point of view, endocytosis has long been regarded as a major mechanism of attenuation, through the degradation of signaling receptors and, in some cases, of their ligands. This outlook has changed, over the past decade, as it has become clear that signaling persists in the endocytic route, and that intracellular endocytic stations (the 'signaling endosomes') actually contribute to the sorting of signals in space and time. Endocytosis-mediated recycling of receptors and of signaling molecules to specific regions of the plasma membrane is also coming into focus as a major mechanism in the execution of spatially restricted functions, such as cell motility. In addition, emerging evidence connects endocytosis as a whole, or individual endocytic proteins, to complex cellular programs, such as the control of the cell cycle, mitosis, apoptosis and cell fate determination. Thus, endocytosis seems to be deeply ingrained into the cell regulation blueprint and its subversion is predicted to play an important role in human diseases: first and foremost, cancer.     

Constitutive high-affinity choline transporter endocytosis is determined by a carboxyl-terminal tail dileucine motif

Maintenance of acetylcholine synthesis depends on the effective functioning of a high-affinity sodium-dependent choline transporter (CHT1). Recent studies have shown that this transporter is predominantly localized inside the cell, unlike other neurotransmitter transporters, suggesting that the trafficking of CHT1 to and from the plasma membrane may play a crucial role in regulating choline uptake. Here we found that CHT1 is rapidly and constitutively internalized in clathrin-coated vesicles to Rab5-positive early endosomes. CHT1 internalization is controlled by an atypical carboxyl-terminal dileucine-like motif (L531, V532) which, upon replacement by alanine residues, blocks CHT1 internalization in both human embryonic kidney 293 cells and primary cortical neurons and results in both increased CHT1 cell surface expression and choline transport activity. Perturbation of clathrin-mediated endocytosis with dynamin-I K44A increases cell surface expression and transport activity to a similar extent as mutating the dileucine motif, suggesting that we have identified the motif responsible for constitutive CHT1 internalization. Based on the observation that the localization of CHT1 to the plasma membrane is transient, we propose that acetylcholine synthesis may be influenced by processes that lead to the attenuation of constitutive CHT1 endocytosis.     

Optical tweezers studies on Notch: single-molecule interaction strength is independent of ligand endocytosis

Notch signaling controls diverse cellular processes critical to development and disease. Cell surface ligands bind Notch on neighboring cells but require endocytosis to activate signaling. The role ligand endocytosis plays in Notch activation has not been established. Here we integrate optical tweezers with cell biological and biochemical methods to test the prevailing model that ligand endocytosis facilitates recycling to enhance ligand interactions with Notch necessary to trigger signaling. Specifically, single-molecule measurements indicate that interference of ligand endocytosis and/or recycling does not alter the force required to rupture bonds formed between cells expressing the Notch ligand Delta-like1 (Dll1) and laser-trapped Notch1 beads. Together, our analyses eliminate roles for ligand endocytosis and recycling in Dll1-Notch1 interactions and indicate that recycling indirectly affects signaling by regulating the accumulation of cell surface ligand. Importantly, our study demonstrates the utility of optical tweezers to test a role for ligand endocytosis in generating cell-mediated mechanical force.     

Ubiquitin-specific Peptidase 8 (USP8) Regulates Endosomal Trafficking of the Epithelial Na+ Channel

Ubiquitination plays a key role in trafficking of the epithelial Na⁺ channel (ENaC). Previous work indicated that ubiquitination enhances ENaC endocytosis and sorting to lysosomes for degradation. Moreover, a defect in ubiquitination causes Liddle syndrome, an inherited form of hypertension. In this work, we identified a role for USP8 in the control of ENaC ubiquitination and trafficking. USP8 increased ENaC current in Xenopus oocytes and collecting duct epithelia and enhanced ENaC abundance at the cell surface in HEK 293 cells. This resulted from altered endocytic sorting; USP8 abolished ENaC degradation in the endocytic pathway, but it had no effect on ENaC endocytosis. USP8 interacted with ENaC, as detected by co-immunoprecipitation, and it deubiquitinated ENaC. Consistent with a functional role for deubiquitination, mutation of the cytoplasmic lysines of ENaC reduced the effect of USP8 on ENaC cell surface abundance. In contrast to USP8, USP2-45 increased ENaC surface abundance by reducing endocytosis but not degradation. Thus, USP8 and USP2-45 selectively modulate ENaC trafficking at different steps in the endocytic pathway. Together with previous work, the data indicate that the ubiquitination state of ENaC is critical for the regulation of epithelial Na⁺ absorption.     

Metabolic depletion of sphingolipids inhibits agonist-induced endocytosis of the serotonin1A receptor

G protein-coupled receptors (GPCRs) are vital cellular signaling machinery and currently represent ~40% drug targets. Endocytosis of GPCRs is an important process that allows stringent spatiotemporal control over receptor population on the cell surface. Although the role of proteins in GPCR endocytosis is well addressed, the contribution of membrane lipids in this process is rather unexplored. Sphingolipids are essential functional lipids in higher eukaryotes and are implicated in several neurological functions. To understand the role of sphingolipids in GPCR endocytosis, we subjected cells expressing human serotonin_1A receptors (an important neurotransmitter GPCR involved in cognitive and behavioral functions) to metabolic sphingolipid depletion using fumonisin B₁, an inhibitor of sphingolipid biosynthetic pathway. Our results, using flow cytometric analysis and confocal microscopic imaging, show that sphingolipid depletion inhibits agonist-induced endocytosis of the serotonin_1A receptor in a concentration-dependent manner, which was restored when sphingolipid levels were replenished. We further show that there was no change in the internalization of transferrin, a marker for clathrin-mediated endocytosis, under sphingolipid-depleted condition, highlighting the specific requirement of sphingolipids for endocytosis of serotonin_1A receptors. Our results reveal the regulatory role of sphingolipids in GPCR endocytosis and highlight the importance of neurotransmitter receptor trafficking in health and disease.     

Fluorescent castasterone reveals BRI1 signaling from the plasma membrane

Receptor-mediated endocytosis is an integral part of signal transduction as it mediates signal attenuation and provides spatial and temporal dimensions to signaling events. One of the best-studied leucine-rich repeat receptor-like kinases in plants, BRASSINOSTEROID INSENSITIVE 1 (BRI1), perceives its ligand, the brassinosteroid (BR) hormone, at the cell surface and is constitutively endocytosed. However, the importance of endocytosis for BR signaling remains unclear. Here we developed a bioactive, fluorescent BR analog, Alexa Fluor 647-castasterone (AFCS), and visualized the endocytosis of BRI1-AFCS complexes in living Arabidopsis thaliana cells. Impairment of endocytosis dependent on clathrin and the guanine nucleotide exchange factor for ARF GTPases (ARF-GEF) GNOM enhanced BR signaling by retaining active BRI1-ligand complexes at the plasma membrane. Increasing the trans-Golgi network/early endosome pool of BRI1-BR complexes did not affect BR signaling. Our findings provide what is to our knowledge the first visualization of receptor-ligand complexes in plants and reveal clathrin- and ARF-GEF-dependent endocytic regulation of BR signaling from the plasma membrane.     

Cholesterol-mediated membrane surface area dynamics in neuroendocrine cells

How cholesterol, a key membrane constituent, affects membrane surface area dynamics in secretory cells is unclear. Using methyl-β-cyclodextrin (MβCD) to deplete cholesterol, we imaged melanotrophs from male Wistar rats in real-time and monitored membrane capacitance (C_m), fluctuations of which reflect exocytosis and endocytosis. Treatment with MβCD reduced cellular cholesterol and caused a dose-dependent attenuation of the Ca²⁺-evoked increase in C_m (IC₅₀ = 5.3 mM) vs. untreated cells. Cytosol dialysis of MβCD enhanced the attenuation of C_m increase (IC₅₀ = 3.3 mM), suggesting cholesterol depletion at intracellular membrane sites was involved in attenuating exocytosis. Acute extracellular application of MβCD resulted in an immediate C_m decline, which correlated well with the cellular surface area decrease, indicating the involvement of cholesterol in the regulation of membrane surface area dynamics. This decline in C_m was three-fold slower than MβCD-mediated fluorescent cholesterol decay, implying that exocytosis is the likely physiological means for plasma membrane cholesterol replenishment. MβCD had no effect on the specific C_m and the blockade of endocytosis by Dyngo 4a, confirmed by inhibition of dextran uptake, also had no effect on the time-course of MβCD-induced C_m decline. Thus acute exposure to MβCD evokes a C_m decline linked to the removal of membrane cholesterol, which cannot be compensated for by exocytosis. We propose that the primary contribution of cholesterol to surface area dynamics is via its role in regulated exocytosis.     

Rapid constitutive internalization and externalization of epidermal growth factor receptors in isolated rat hepatocytes. Monensin inhibits receptor externalization and reduces the capacity for continued endocytosis of epidermal growth factor

It was previously demonstrated that freshly isolated rat hepatocytes can internalize severalfold more epidermal growth factor (EGF) molecules than the number of surface EGF receptors, suggesting extensive reutilization of receptors during endocytosis (Gladhaug, I. P. & Christoffersen, T. (1987) Eur. J. Biochem. 164, 267-275). The present report attempts to explore the pathways involved in the externalization of EGF receptors. Incubation of hepatocytes at 37 degrees C in the absence of ligand increased the surface receptor pool by 50-100% within 45 min. Pretreatment with monensin inhibited the turnover of the surface EGF receptor pool by 50-60% within 10 min and blocked the temperature-dependent externalization of receptors. Cycloheximide caused a slower attenuation of the surface receptor pool, whereas tunicamycin and chloroquine did not significantly affect the exchange of receptor pools. Monensin reduced the surface receptor pool and the endocytic uptake in corresponding proportions, without affecting the internalization of prebound EGF. Endocytic uptake was unaffected by chloroquine and slightly reduced by cycloheximide. The internalization of unoccupied receptors and the endocytosis of prebound EGF followed similar kinetics (t1/2 approximately 5 min), suggesting that unoccupied receptors are internalized at a rate comparable to that of occupied receptors. The results suggest that there is a rapid turnover of the surface pool of EGF receptors with constitutive internalization of unoccupied surface receptors and externalization of internal receptors. This is consistent with, but does not prove, a true recycling of the EGF receptors in the hepatocytes. The monensin-sensitive externalization pathway determines the capacity for continued endocytosis of EGF.     

Dpp gradient formation by dynamin-dependent endocytosis: receptor trafficking and the diffusion model

Developing cells acquire positional information by reading the graded distribution of morphogens. In Drosophila, the Dpp morphogen forms a long-range concentration gradient by spreading from a restricted source in the developing wing. It has been assumed that Dpp spreads by extracellular diffusion. Under this assumption, the main role of endocytosis in gradient formation is to downregulate receptors at the cell surface. These surface receptors bind to the ligand and thereby interfere with its long-range movement. Recent experiments indicate that Dpp spreading is mediated by Dynamin-dependent endocytosis in the target tissue, suggesting that extracellular diffusion alone cannot account for Dpp dispersal. Here, we perform a theoretical study of a model for morphogen spreading based on extracellular diffusion, which takes into account receptor binding and trafficking. We compare profiles of ligand and surface receptors obtained in this model with experimental data. To this end, we monitored directly the pool of surface receptors and extracellular Dpp with specific antibodies. We conclude that current models considering pure extracellular diffusion cannot explain the observed role of endocytosis during Dpp long-range movement.     

Nedd4-2 induces endocytosis and degradation of proteolytically cleaved epithelial Na+ channels

As a pathway for Na(+) reabsorption, the epithelial Na(+) channel ENaC is critical for Na(+) homeostasis and blood pressure control. Na(+) transport is regulated by Nedd4-2, an E3 ubiquitin ligase that decreases ENaC expression at the cell surface. To investigate the underlying mechanisms, we proteolytically cleaved/activated ENaC at the cell surface and then quantitated the rate of disappearance of cleaved channels using electrophysiological and biochemical assays. We found that cleaved ENaC channels were rapidly removed from the cell surface. Deletion or mutation of the Nedd4-2 binding motifs in alpha, beta, and gammaENaC dramatically reduced endocytosis, whereas a mutation that disrupts a YXX? endocytosis motif had no effect. ENaC endocytosis was also decreased by silencing of Nedd4-2 and by expression of a dominant negative Nedd4-2 construct. Conversely, Nedd4-2 overexpression increased ENaC endocytosis in human embryonic kidney 293 cells but had no effect in Fischer rat thyroid epithelia. In addition to its effect on endocytosis, Nedd4-2 also increased the rate of degradation of the cell surface pool of cleaved alphaENaC. Together the data indicate that Nedd4-2 reduces ENaC surface expression by altering its trafficking at two distinct sites in the endocytic pathway, inducing endocytosis of cleaved channels and targeting them for degradation.     

Phosphorylated ITIMs enable ubiquitylation of an inhibitory cell surface receptor

Immune responses are modulated by activating and inhibitory receptors that traffic to and from the cell surface. Ligands that bind to inhibitory receptors induce phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic tails, followed by recruitment of inhibitory signaling molecules. Mechanisms that control the surface levels of inhibitory receptors are largely unexplored. Here, we show, using CD33/sialic acid-binding immunoglobulin-like lectin (Siglec)-3 as a paradigm, that ITIMs can bind to the ubiquitin ligase Cbl and that ITIMs are ubiquitylated following Src family kinase-mediated tyrosine phosphorylation. Ubiquitylation is a known signal for endocytosis. Accordingly, cells expressing CD33 mutants that cannot become ubiquitylated show significantly increased cell surface expression of CD33 and have impaired CD33 internalization, whereas in-frame fusion of ubiquitin to CD33 reverses this phenotype. Our results identify a novel function of ITIMs and demonstrate that phosphorylation-dependent ubiquitylation regulates cell surface expression and internalization, and thus possibly function, of CD33/Siglec-3, suggesting an important role of ubiquitin in endocytosis of ITIM-bearing inhibitory immunoreceptors.     

Small interfering RNA profiling reveals key role of clathrin-mediated endocytosis and early endosome formation for infection by respiratory syncytial virus

Respiratory syncytial virus (RSV) is a common cause of respiratory tract infections in infants and the elderly. Like many other pH-independent enveloped viruses, RSV is thought to enter at the cell surface, independently of common endocytic pathways. We have used a targeted small interfering RNA (siRNA) library to identify key cellular genes involved in cytoskeletal dynamics and endosome trafficking that are important for RSV infection. Surprisingly, RSV infection was potently inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis, including clathrin light chain. The important role of clathrin-mediated endocytosis was confirmed by the expression of well-characterized dominant-negative mutants of genes in this pathway and by using the clathrin endocytosis inhibitor chlorpromazine. We conclude that, while RSV may be competent to enter at the cell surface, clathrin function and endocytosis are a necessary and important part of a productive RSV infection, even though infection is strictly independent of pH. These findings raise the possibility that other pH-independent viruses may share a similar dependence on endocytosis for infection and provide a new potential avenue for treatment of infection.     

A dynamic actin cytoskeleton functions at multiple stages of clathrin-mediated endocytosis

Clathrin-mediated endocytosis in mammalian cells is critical for a variety of cellular processes including nutrient uptake and cell surface receptor down-regulation. Despite the findings that numerous endocytic accessory proteins directly or indirectly regulate actin dynamics and that actin assembly is spatially and temporally coordinated with endocytosis, direct functional evidence for a role of actin during clathrin-coated vesicle formation is lacking. Here, we take parallel biochemical and microscopic approaches to address the contribution of actin polymerization/depolymerization dynamics to clathrin-mediated endocytosis. When measured using live-cell fluorescence microscopy, disruption of the F-actin assembly and disassembly cycle with latrunculin A or jasplakinolide results in near complete cessation of all aspects of clathrin-coated structure (CCS) dynamics. Stage-specific biochemical assays and quantitative fluorescence and electron microscopic analyses establish that F-actin dynamics are required for multiple distinct stages of clathrin-coated vesicle formation, including coated pit formation, constriction, and internalization. In addition, F-actin dynamics are required for observed diverse CCS behaviors, including splitting of CCSs from larger CCSs, merging of CCSs, and lateral mobility on the cell surface. Our results demonstrate a key role for actin during clathrin-mediated endocytosis in mammalian cells.     

Increased internalization of p120-uncoupled E-cadherin and a requirement for a dileucine motif in the cytoplasmic domain for endocytosis of the protein

E-cadherin is a member of the cadherin family of Ca2+-dependent cell-cell adhesion molecules. E-cadherin associates with beta-catenin at the membrane-distal region of its cytosolic domain and with p120 at the membrane-proximal region of its cytoplasmic domain. It has been shown that a pool of cell surface E-cadherin is constitutively internalized and recycled back to the surface. Further, p120 knockdown by small interference RNA resulted in dose-dependent elimination of cell surface E-cadherin. Consistent with these observations, we found that selective uncoupling of p120 from E-cadherin by introduction of amino acid substitutions in the p120-binding site increased the level of E-cadherin endocytosis. The increased endocytosis was clathrin-dependent, because it was blocked by expression of a dominant-negative form of dynamin or by hypertonic shock. A dileucine motif in the juxtamembrane cytoplasmic domain is required for E-cadherin endocytosis, because substitution of these residues to alanine resulted in impaired internalization of the protein. The alanine substitutions in the p120-uncoupled construct reduced endocytosis of the protein, indicating that this motif was dominant to p120 binding in the control of E-cadherin endocytosis. Therefore, these results are consistent with the idea that p120 regulates E-cadherin endocytosis by masking the dileucine motif and preventing interactions with adaptor proteins required for internalization.     

A role of the sulfonylurea receptor 1 in endocytic trafficking of ATP-sensitive potassium channels

The ATP-sensitive potassium (K(ATP) ) channel consisting of sulfonylurea receptor 1 (SUR1) and inward-rectifier potassium channel 6.2 (Kir6.2) has a well-established role in insulin secretion. Mutations in either subunit can lead to disease due to aberrant channel gating, altered channel density at the cell surface or a combination of both. Endocytic trafficking of channels at the plasma membrane is one way to influence surface channel numbers. It has been previously reported that channel endocytosis is dependent on a tyrosine-based motif in Kir6.2, while SUR1 alone is unable to internalize. In this study, we followed endocytic trafficking of surface channels in real time by live-cell imaging of channel subunits tagged with an extracellular minimal α-bungarotoxin-binding peptide labeled with a fluorescent dye. We show that SUR1 undergoes endocytosis independent of Kir6.2. Moreover, mutations in the putative endocytosis motif of Kir6.2, Y330C, Y330A and F333I are unable to prevent channel endocytosis. These findings challenge the notion that Kir6.2 bears the sole endocytic signal for K(ATP) channels and support a role of SUR1 in this trafficking process.     

Suppressors of T-cell receptor signaling Sts-1 and Sts-2 bind to Cbl and inhibit endocytosis of receptor tyrosine kinases

The ubiquitin (Ub) ligase Cbl plays a critical role in attenuation of receptor tyrosine kinase (RTK) signaling by inducing ubiquitination of RTKs and promoting their sorting for endosomal degradation. Herein, we describe the identification of two novel Cbl-interacting proteins, p70 and Clip4 (recently assigned the names Sts-1 and Sts-2, respectively), that inhibit endocytosis of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor. Sts-1 and Sts-2 contain SH3 domains that interacted with Cbl, Ub-associated domains, which bound directly to mono-Ub or to the EGFR/Ub chimera as well as phosphoglycerate mutase domains that mediated oligomerization of Sts-1/2. Ligand-induced recruitment of Sts-1/Sts-2 into activated EGFR complexes led to inhibition of receptor internalization, reduction in the number of EGFR-containing endocytic vesicles, and subsequent block of receptor degradation followed by prolonged activation of mitogenic signaling pathways. On the other hand, interference with Sts-1/Sts-2 functions diminished ligand-induced receptor degradation, cell proliferation, and oncogenic transformation in cultured fibroblasts. We suggest that Sts-1 and Sts-2 represent a novel class of Ub-binding proteins that regulate RTK endocytosis and control growth factor-induced cellular functions.