The Initiation of Clathrin-Mediated Endocytosis Is Mechanistically Highly Flexible

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Inhibition of Clathrin-Mediated Endocytosis Selectively Attenuates Specific Insulin Receptor Signal Transduction Pathways

To examine the role of clathrin-dependent insulin receptor internalization in insulin-stimulated signal transduction events, we expressed a dominant-interfering mutant of dynamin (K44A/dynamin) by using a recombinant adenovirus in the H4IIE hepatoma and 3T3L1 adipocyte cell lines. Expression of K44A/dynamin inhibited endocytosis of the insulin receptor as determined by both cell surface radioligand binding and trypsin protection analysis. The inhibition of the insulin receptor endocytosis had no effect on either the extent of insulin receptor autophosphorylation or insulin receptor substrate 1 (IRS1) tyrosine phosphorylation. In contrast, expression of K44A/dynamin partially inhibited insulin-stimulated Shc tyrosine phosphorylation and activation of the mitogen-activated protein kinases ERK1 and -2. Although there was an approximately 50% decrease in the insulin-stimulated activation of the phosphatidylinositol 3-kinase associated with IRS1, insulin-stimulated Akt kinase phosphorylation and activation were unaffected. The expression of K44A/dynamin increased the basal rate of amino acid transport, which was additive with the effect of insulin but had no effect on the basal or insulin-stimulated DNA synthesis. In 3T3L1 adipocytes, expression of K44A/dynamin increased the basal rate of glucose uptake, glycogen synthesis, and lipogenesis without any significant effect on insulin stimulation. Together, these data demonstrate that the acute actions of insulin are largely independent of insulin receptor endocytosis and are initiated by activation of the plasma membrane-localized insulin receptor.     

Cell Migration Is Regulated by Platelet-Derived Growth Factor Receptor Endocytosis

Cell migration requires spatial and temporal processes that detect and transfer extracellular stimuli into intracellular signals. The platelet-derived growth factor (PDGF) receptor is a cell surface receptor on fibroblasts that regulates proliferation and chemotaxis in response to PDGF. How the PDGF signal is transmitted accurately through the receptor into cells is an unresolved question. Here, we report a new intracellular signaling pathway by which DOCK4, a Rac1 guanine exchange factor, and Dynamin regulate cell migration by PDGF receptor endocytosis. We showed by a series of biochemical and microscopy techniques that Grb2 serves as an adaptor protein in the formation of a ternary complex between the PDGF receptor, DOCK4, and Dynamin, which is formed at the leading edge of cells. We found that this ternary complex regulates PDGF-dependent cell migration by promoting PDGF receptor endocytosis and Rac1 activation at the cell membrane. This study revealed a new mechanism by which cell migration is regulated by PDGF receptor endocytosis.Chemoattractants bind to cell surface receptors, resulting in the cytoskeletal reorganization that permits the migration of cells toward a stimulus. In fibroblasts, the platelet-derived growth factor receptor β (PDGFRβ) is a cell surface receptor tyrosine kinase (RTK) that regulates cell proliferation and chemotaxis in response to PDGF. PDGF binding activates PDGF receptor autophosphorylation, which in turn mediates a series of intracellular signaling cascades initiated by the association of SH2 domain-containing adaptor proteins (25). The adaptor protein Grb2 at the plasma membrane binds to Ras exchange factor Sos1, activating mitogen-activated protein kinase (MAPK) and cell proliferation signals (19). Grb2 also plays a critical role in receptor internalization via its interaction with dynamin, an exchange factor that facilitates receptor entry into endocytic vesicles (32). Grb2 regulates ubiquitination and the degradation of the receptor via its interaction with Cbl, an E3 ubiquitin ligase (33). While the role of Grb2 in modulating receptor levels and facilitating growth factor-dependent mitogenic signals is defined, its role in coordinating receptor-dependent chemotaxis has not been elucidated.The small GTPase Rac1 plays a crucial role in PDGF-mediated chemotaxis by regulating cortical actin at the leading edge of cells. PDGF receptor activation promotes GTP loading and the translocation of Rac1 to the cell membrane via guanine exchange factors (GEFs). The DOCK family of Rac1 GEFs, also called CDM proteins (for Caenorhabditis elegans ced-5, vertebrate DOCK180, and Drosophila myoblast city), are regulators of cell migration and have been implicated in various biological processes, such as lymphocyte migration, phagocytosis, and cancer progression (6, 10, 30, 35). In migrating fibroblasts, DOCK proteins localize to the cell''s leading edge via their interaction with the phospholipid PIP3, but a direct molecular link to PDGF has not been established (5). Biochemical studies show that Rac activation requires the DHR2/docker domain of DOCK proteins and the expression of the PH domain-containing protein Ced-12/ELMO. Previously we identified DOCK4 in a screen for novel tumor suppressor genes using representational difference analysis on mouse tumor cell lines (35). DOCK4, like other CDM proteins, binds ELMO and exerts its biochemical effects on the small GTPases Rac and Rap1 (30, 35). An interesting observation is that the amino acid sequence toward the C terminus is not conserved among individual DOCK family members. The alternate splicing of the DOCK4 gene has been reported, but how amino acid sequence variation alters the signaling properties of DOCK4 for the regulation of cell migration is unknown.Members of the Nck family of adaptor proteins, CrkII and Nck, have been reported to bind to the C terminus of DOCK180 (12, 29). Here, we show that the third member of the family of Nck adaptors, namely Grb2, binds to wild-type DOCK4. We found that a ternary complex formed by Grb2-DOCK4-Dynamin2 interacts with PDGF-activated PDGFβ receptor and promotes growth factor-dependent migration without altering cell proliferation. PDGF-dependent migration requires receptor endocytosis and is regulated by the formation of a DOCK4-Grb2-Dynamin2-PDGFRβ complex at the cell''s leading edge. These studies provide novel mechanistic insights into PDGFRβ regulation and cell migration.     

Cathepsin X Cleaves Profilin 1 C-Terminal Tyr139 and Influences Clathrin-Mediated Endocytosis

Cathepsin X, a cysteine carboxypeptidase, is upregulated in several types of cancer. Its molecular target in tumor cells is profilin 1, a known tumor suppressor and regulator of actin cytoskeleton dynamics. Cathepsin X cleaves off the C-terminal Tyr139 of profilin 1, affecting binding of poly-L-proline ligands and, consequently, tumor cell migration and invasion. Profilin 1 with mutations at the C-terminus, transiently expressed in prostate cancer cells PC-3, showed that Tyr139 is important for proper function of profilin 1 as a tumor suppressor. Cleaving off Tyr139 prevents the binding of clathrin, a poly-L-proline ligand involved in endocytosis. More profilin 1—clathrin complexes were present in PC-3 cells when cathepsin X was inhibited by its specific inhibitor AMS36 or silenced by siRNA. As a consequence, the endocytosis of FITC-labeled dextran and transferrin conjugate was significantly increased. These results constitute the first report of the regulation of clathrin-mediated endocytosis in tumor cells through proteolytic processing of profilin 1.     

Uptake of Rabies Virus into Epithelial Cells by Clathrin-Mediated Endocytosis Depends upon Actin

Rabies virus (RABV) causes a fatal zoonotic encephalitis. Disease symptoms require replication and spread of the virus within neuronal cells; however, in infected animals as well as in cell culture the virus replicates in a broad range of cell types. Here we use a single-cycle RABV and a recombinant vesicular stomatitis virus (rVSV) in which the glycoprotein (G) was replaced with that of RABV (rVSV RABV G) to examine RABV uptake into the African green monkey kidney cell line BS-C-1. Combining biochemical studies and real-time spinning-disk confocal fluorescence microscopy, we show that the predominant entry pathway of RABV particles into BS-C-1 cells is clathrin dependent. Viral particles enter cells in pits with elongated structures and incomplete clathrin coats which depend upon actin to complete the internalization process. By measuring the time of internalization and the abundance of the clathrin adaptor protein AP2, we further show that the pits that internalize RABV particles are similar to those that internalize VSV particles. Pharmacological perturbations of dynamin or of actin polymerization inhibit productive infection, linking our observations on particle uptake with viral infectivity. This work extends to RABV particles the finding that clathrin-mediated endocytosis of rhabdoviruses proceeds through incompletely coated pits which depend upon actin.     

New Regulators of Clathrin-Mediated Endocytosis Identified in Saccharomyces cerevisiae by Systematic Quantitative Fluorescence Microscopy

Despite the importance of clathrin-mediated endocytosis (CME) for cell biology, it is unclear if all components of the machinery have been discovered and many regulatory aspects remain poorly understood. Here, using Saccharomyces cerevisiae and a fluorescence microscopy screening approach we identify previously unknown regulatory factors of the endocytic machinery. We further studied the top scoring protein identified in the screen, Ubx3, a member of the conserved ubiquitin regulatory X (UBX) protein family. In vivo and in vitro approaches demonstrate that Ubx3 is a new coat component. Ubx3-GFP has typical endocytic coat protein dynamics with a patch lifetime of 45 ± 3 sec. Ubx3 contains a W-box that mediates physical interaction with clathrin and Ubx3-GFP patch lifetime depends on clathrin. Deletion of the UBX3 gene caused defects in the uptake of Lucifer Yellow and the methionine transporter Mup1 demonstrating that Ubx3 is needed for efficient endocytosis. Further, the UBX domain is required both for localization and function of Ubx3 at endocytic sites. Mechanistically, Ubx3 regulates dynamics and patch lifetime of the early arriving protein Ede1 but not later arriving coat proteins or actin assembly. Conversely, Ede1 regulates the patch lifetime of Ubx3. Ubx3 likely regulates CME via the AAA-ATPase Cdc48, a ubiquitin-editing complex. Our results uncovered new components of the CME machinery that regulate this fundamental process.     

Characterization of the Kremen-binding site on Dkk1 and elucidation of the role of Kremen in Dkk-mediated Wnt antagonism

Wnt signaling is involved in a wide range of developmental, physiological, and pathophysiological processes and is negatively regulated by Dickkopf1 (Dkk1). Dkk1 has been shown to bind to two transmembrane proteins, the low density lipoprotein receptor-related proteins (LRP) 5/6 and Kremen. Here, we show that Dkk1 residues Arg(197), Ser(198), and Lys(232) are specifically involved in its binding to Kremen rather than to LRP6. These residues are localized at a surface that is at the opposite side of the LRP6-binding surface based on a three-dimensional structure of Dkk1 deduced from that of Dkk2. We were surprised to find that the Dkk1 mutants carrying a mutation at Arg(197), Ser(198), or Lys(232), the key Kremen-binding residues, could antagonize Wnt signaling as well as the wild-type Dkk1. These mutations only affected their ability to antagonize Wnt signaling when both LRP6 and Kremen were coexpressed. These results suggest that Kremen may not be essential for Dkk1-mediated Wnt antagonism and that Kremen may only play a role when cells express a high level of LRP5/6.     

Estrogen Receptor Alpha Is Expressed in Mesenteric Mesothelial Cells and Is Internalized in Caveolae upon Freund's Adjuvant Treatment

Transformation of epithelial cells into connective tissue cells (epithelial-mesenchymal transition, EMT) is a complex mechanism involved in tumor metastasis, and in normal embryogenesis, while type II EMT is mainly associated with inflammatory events and tissue regenaration.In this study we examined type II EMT at the ultrastructural and molecular level during the inflammatory process induced by Freund''s adjuvant treatment in rat mesenteric mesothelial cells. We found that upon the inflammatory stimulus mesothelial cells lost contact with the basal lamina and with each other, and were transformed into spindle-shaped cells. These morphological changes were accompanied by release of interleukins IL-1alpha, -1beta and IL-6 and by secretion of transforming growth factor beta (TGF-β) into the peritoneal cavity. Mesothelial cells also expressed estrogen receptor alpha (ER-α) as shown by immunolabeling at the light and electron microscopical levels, as well as by quantitative RT-PCR. The mRNA level of ER-α showed an inverse correlation with the secretion of TGF-β. At the cellular and subcellular levels ER-α was colocalized with the coat protein caveolin-1 and was found in the plasma membrane of mesothelial cells, in caveolae close to multivesicular bodies (MVBs) or in the membrane of these organelles, suggesting that ER-α is internalized via caveola-mediated endocytosis during inflammation. We found asymmetric, thickened, electron dense areas on the limiting membrane of MVBs (MVB plaques) indicating that these sites may serve as platforms for collecting and organizing regulatory proteins. Our morphological observations and biochemical data can contribute to form a potential model whereby ER-α and its caveola-mediated endocytosis might play role in TGF-β induced type II EMT in vivo.     

GM1 Ganglioside Activates the High-Affinity Nerve Growth Factor Receptor trkA

Abstract: The monosialoganglioside GM1 has been shown to possess neurotrophic activity in vitro and in vivo and is now used as an experimental treatment for a variety of neurological disorders and trauma. Little is known about the mechanism of action used by GM1. Because GM1 appears to enhance nerve growth factor (NGF) activity, we have used C6trk⁺ cells, a derivative of C6-2B glioma cells that express the high-affinity receptor for NGF trkA , to determine whether the neurotrophic effects of GM1 occurs through induction of trkA activity. Exposure of C6trk⁺ cells to NGF (10–50 ng/ml) resulted in a five- to 10-fold increase in trkA tyrosine phosphorylation within 5 min. Incubation of cells with GM1 resulted in a threefold increase in trkA phosphorylation beginning within 1 h and peaking between 3 and 6 h. Optimal responses to GM1 were obtained using 80–100 µ M concentrations. Moreover, tyrosine phosphorylation of known trkA target proteins, such as extracellular signal-regulated kinases, and suc -associated neurotrophic factor-induced tyrosine-phosphorylated target, were activated upon stimulation of C6trk⁺ cells with GM1. In addition, GM1 potentiated the NGF-mediated activation of tyrosine phosphorylation of trkA . GM1 failed to induce phosphorylation of trkA and target proteins in mock transfected cells. Thus, our data demonstrate that GM1 mimics some of the effects of NGF and suggest that the neurotrophic properties of GM1 may be attributed to its activation of trkA signal transduction.     

Kinin-Stimulated B1 Receptor Signaling Depends on Receptor Endocytosis Whereas B2 Receptor Signaling Does Not

Kinins are potent pro-inflammatory peptides that act through two G protein-coupled receptor subtypes, B1 (B1R) and B2 (B2R). Kinin-stimulated B2R signaling is often transient, whereas B1R signaling is sustained. This was confirmed by monitoring agonist-stimulated intracellular Ca²⁺ mobilization in A10 smooth muscle cells expressing human wild-type B2R and B1R. We further studied the role of receptor membrane trafficking in receptor-mediated phosphoinositide (PI) hydrolysis in model HEK293 cell lines stably expressing the receptors. Treatment of cells with brefeldin A, to inhibit maturation of de novo synthesized receptors, or hypertonic sucrose, to inhibit receptor endocytosis, showed that the basal cell surface receptor turnover was considerably faster for B1R than for B2R. Inhibition of endocytosis, which stabilized B1R on the cell surface, inhibited B1R signaling, whereas B2R signaling was not perturbed. Signaling by a B1R construct in which the entire C-terminal domain was deleted remained sensitive to inhibition of receptor endocytosis, whereas signaling by a B1R construct in which this domain was substituted with the corresponding domain in B2R was not sensitive. B2R and B1R co-expression, which also appeared to stabilize B1R on the cell surface, presumably by receptor hetero-dimerization, also inhibited B1R signaling, whereas B2R signaling was slightly enhanced. Furthermore, the B2R-specific agonist bradykinin (BK) directed both receptors through a common endocytic pathway, whereas the B1R-specific agonist Lys-desArg⁹-BK was unable to do so. These results suggest that B1R-mediated PI hydrolysis depends on a step in receptor endocytosis, whereas B2R-mediated PI hydrolysis does not. We propose that B1R uses at least part of the endocytic machinery to sustain agonist-promoted signaling.     

NECAP 1 Regulates AP-2 Interactions to Control Vesicle Size,Number, and Cargo During Clathrin-Mediated Endocytosis

AP-2 is the core-organizing element in clathrin-mediated endocytosis. During the formation of clathrin-coated vesicles, clathrin and endocytic accessory proteins interact with AP-2 in a temporally and spatially controlled manner, yet it remains elusive as to how these interactions are regulated. Here, we demonstrate that the endocytic protein NECAP 1, which binds to the α-ear of AP-2 through a C-terminal WxxF motif, uses an N-terminal PH-like domain to compete with clathrin for access to the AP-2 β2-linker, revealing a means to allow AP-2–mediated coordination of accessory protein recruitment and clathrin polymerization at sites of vesicle formation. Knockdown and functional rescue studies demonstrate that through these interactions, NECAP 1 and AP-2 cooperate to increase the probability of clathrin-coated vesicle formation and to control the number, size, and cargo content of the vesicles. Together, our data demonstrate that NECAP 1 modulates the AP-2 interactome and reveal a new layer of organizational control within the endocytic machinery.     

Desensitization of the Neurokinin 1 Receptor Is Mediated by the Receptor Carboxy-Terminal Region, but Is Not Caused by Receptor Internalization

Abstract: The carboxy-terminal cytoplasmic regions of the rat neurokinin 1 (substance P) and neurokinin 2 (neurokinin A) receptors have been exchanged to determine if this region of the neurokinin 1 receptor is involved in its desensitization. When expressed at similar levels in stably transfected Chinese hamster ovary (CHO) cell lines, receptors containing the carboxy-terminal region of the neurokinin 1 receptor desensitized significantly more (as measured by reduction of the inositol 1,4,5-trisphosphate response) when preexposed for 1 min to 1 µ M neurokinin, indicating a role for the carboxy-terminal region of the neurokinin 1 receptor in its desensitization. Measurement of receptor internalization using radiolabeled neurokinins (0.3 n M ) indicated that ∼75–80% of the receptors were internalized in each cell line after 10 min at 37°C, with no observable correlation between neurokinin receptor desensitization and internalization. Measurement of loss of receptor surface sites for cell lines CHO NK1 and CHO NK1NK2 following exposure to 1 µ M substance P also indicated no obvious relationship between the percent desensitization and percent of receptors internalized. Also, two inhibitors of neurokinin 1 receptor internalization, phenylarsine oxide and hyperosmolar sucrose, did not inhibit neurokinin 1 receptor desensitization. The protein kinase inhibitors Ro 31-8220, staurosporine, and Zn²⁺ had no effect on neurokinin 1 receptor desensitization, indicating that the kinases affected by these agents are not rate-limiting in neurokinin 1 receptor desensitization in this system.     

WNT Activates the AAK1 Kinase to Promote Clathrin-Mediated Endocytosis of LRP6 and Establish a Negative Feedback Loop

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Urokinase-Type Plasminogen Activator Receptor Is Internalized by Different Mechanisms in Polarized and Nonpolarized Madin–Darby Canine Kidney Epithelial Cells

Accumulated data indicate that endocytosis of the glycosylphosphatidyl-inositol-anchored protein urokinase plasminogen activator receptor (uPAR) depends on binding of the ligand uPA:plasminogen activator inhibitor-1 (PAI-1) and subsequent interaction with internalization receptors of the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP). We show that uPAR with bound uPA:PAI-1 is capable of entering cells in a clathrin-independent process. First, HeLa^K44A cells expressing mutant dynamin efficiently internalized uPA:PAI-1 under conditions in which transferrin endocytosis was blocked. Second, in polarized Madin–Darby canine kidney (MDCK) cells, which expressed human uPAR apically, the low basal rate of uPAR ligand endocytosis, which could not be inhibited by RAP, was increased by forskolin or phorbol ester (phorbol 12-myristate 13-acetate), which selectively up-regulate clathrin-independent endocytosis from the apical domain of epithelial cells. Third, in subconfluent nonpolarized MDCK cells, endocytosis of uPA:PAI-1 was only decreased marginally by RAP. At the ultrastructural level uPAR was largely excluded from clathrin-coated pits in these cells and localized in invaginated caveolae only in the presence of cross-linking antibodies. Interestingly, a larger fraction of uPAR in nonpolarized relative to polarized MDCK cells was insoluble in Triton X-100 at 0°C, and by surface labeling with biotin we also show that internalized uPAR was mainly detergent insoluble, suggesting a correlation between association with detergent-resistant membrane microdomains and higher degree of clathrin-independent endocytosis. Furthermore, by cryoimmunogold labeling we show that 5–10% of internalized uPAR in nonpolarized, but not polarized, MDCK cells is targeted to lysosomes by a mechanism that is regulated by ligand occupancy.     

Constitutive and Regulated Endocytosis of the Glycine Transporter GLYT1b Is Controlled by Ubiquitination

The glycine transporter GLYT1 regulates both glycinergic and glutamatergic neurotransmission by controlling the reuptake of glycine at synapses. Trafficking of GLYT1 to and from the cell surface is critical for its function. Activation of PKC down-regulates the activity of GLYT1 through a mechanism that has so far remained uncharacterized. Here we show that GLYT1b undergoes fast constitutive endocytosis that is accelerated by phorbol esters. Both constitutive and regulated endocytosis occur through a dynamin 2- and clathrin-dependent pathway, accumulating in the transporter in transferrin-containing endosomes. A chimera with the extracellular and transmembrane domains of the nerve growth factor receptor and the COOH-terminal tail of GLYT1 was efficiently internalized through this clathrin pathway, suggesting the presence of molecular determinants for GLYT1b endocytosis in its COOH-terminal tail. Extensive site-directed mutagenesis in this region of the chimera highlighted the involvement of lysine residues in its internalization. In the context of the full-length transporter, lysine 619 played a prominent role in both the constitutive and phorbol 12-myristate 13-acetate-induced endocytosis of GLYT1b, suggesting the involvement of ubiquitin modification of GLYT1b during the internalization process. Indeed, we show that GLYT1b undergoes ubiquitination and that this process is stimulated by phorbol 12-myristate 13-acetate. In addition, this endocytosis is impaired in an ubiquitination-deficient cell line, further evidence that constitutive and regulated endocytosis of GLYT1b is ubiquitin-dependent. It remains to be determined whether GLYT1b recycling might be affected in pathologies involving alterations to the ubiquitin system, thereby interfering with its influence on inhibitory and excitatory neurotransmission.Glycine fulfills a dual role in neurotransmission by mediating inhibition through the strychnine-sensitive glycine receptor and excitation as a co-agonist of the NMDA² receptors (1, 2). Although it was initially believed that the concentration of glycine in the synaptic cleft would be sufficient to saturate the glycine sites on NMDA receptors, recent pharmacological and electrophysiological evidence indicates that due to the activity of the GLYT1 (glycine transporter-1) glycine transporter, this is probably not the case. Three isoforms of GLYT1 exist that differ in their NH₂-terminal sequence (GLYT1a, GLYT1b, and GLYT1c), and they are strongly expressed in glycinergic areas of the nervous system, predominantly in glial cells (3). Indeed, mice lacking GLYT1 have impaired glycinergic neurotransmission, which has been attributed to an increase in extracellular glycine close to the strychnine-sensitive glycine receptor (4). Moreover, GLYT1 has been identified in neuronal elements closely associated with the glutamatergic pathways throughout the brain (5, 6). GLYT1 is enriched in presynaptic buttons, where it largely co-localizes with the vesicular glutamate transporter vGLUT1. It is also present in the postsynaptic densities of asymmetric synapses, and complexes containing both NMDA receptor and GLYT1 have been shown to exist (5). In these postsynaptic sites, the distribution of GLYT1 is partially controlled through its interaction with the scaffolding protein PSD-95 (7). Accordingly, GLYT1 is believed to play a role in controlling the concentration of glycine in the microenvironment around the NMDA receptor. Indeed, functional studies have shown that a specific GLYT1 inhibitor, N-[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine, potentiates NMDA-mediated responses in vitro and in vivo (8–10). The potential role of GLYT1 in glutamatergic neurotransmission has also been confirmed in heterozygous Glyt1^+/− animals that express only 50% of the normal levels of GLYT1 as well as when GLYT1 expression is disturbed in forebrain neurons. In these animals, hippocampal NMDA receptor function is enhanced, and the mice appear to display better memory retention than wild type mice (11–13).The mechanisms responsible for the insertion of GLYT1 into glutamatergic synapses are unknown. However, recent studies indicate that the movement of transporters within the cell is highly organized and that a number of ancillary proteins control their intracellular trafficking by interacting with targeting motifs in the transporter. Indeed, like other neurotransmitter transporters, GLYT1 is asymmetrically distributed in polarized cells (14, 15). The asymmetric distribution of sodium-dependent neurotransmitter transporters (NSS) requires a number of steps that commence with their efficient exit from the endoplasmic reticulum. This is followed by sorting processes in the Golgi complex, insertion into the plasma membrane, and the retention of the transporter at functional synaptic sites. Moreover, the amount of transporter in the plasma membrane is also regulated by endocytosis and recycling mechanisms. Like several other members of the NSS family, GLYT1 is subjected to regulation by protein kinase C. Activation of PKC by phorbol esters down-regulates GLYT1, which is endocytosed from the plasma membrane to intracellular compartments in several cell lines (16–18). For years, the molecular mechanisms that mediate phorbol 12-myristate 13-acetate (PMA)-stimulated endocytosis of the NSS family members have remained elusive. However, recent evidence obtained for the dopamine transporter (DAT) has revealed the importance of ubiquitination of DAT for its endocytosis (19). DAT is ubiquitinated by the Nedd4-2 ligase at several intracellular lysines. Indeed, mutation of these lysines abolished both ubiquitination and phorbol ester-stimulated endocytosis, indicating that the associated ubiquitin molecules serve as a platform to recruit endocytotic adaptors (20–23). Ubiquitination has also been implicated in the endocytosis of other membrane proteins, including the main transporter for glutamate, GLT1, and the system A transporter SNAT2 (24–26).Ubiquitin coupling can involve either mono- or polyubiquitination. Monoubiquitination occurs when a single ubiquitin molecule is coupled to one or more lysine residues on a target protein, such that the final stoichiometry is one ubiquitin per lysine. Polyubiquitination refers to the coupling of a chain of ubiquitins to a lysine on the target protein, with a final stoichiometry of four or more ubiquitins per lysine. Whereas monoubiquitinated proteins are degraded in lysosomes, polyubiquitinated proteins are recognized by and subsequently degraded by the 26 S proteasome (27).In this study, we show that GLYT1b is endocytosed through a clathrin-dependent mechanism, a process that is accelerated by phorbol esters. Through a mutational analysis, we have identified a lysine residue in the COOH-terminal tail of the protein as the major determinant for GLYT1b internalization through both constitutive and PMA-stimulated pathways. Ubiquitination GLYT1b is stimulated by PMA, a finding compatible with ubiquitin being the platform on which the clathrin network is assembled.