Clathrin-dependent and independent endocytosis of glucose transporter 4 (GLUT4) in myoblasts: regulation by mitochondrial uncoupling

In myocytes and adipocytes, insulin increases glucose transporter 4 (GLUT4) exocytosis by promoting GLUT4 vesicle docking/fusion with the membrane. Less is known about the mechanism and regulation of GLUT4 endocytosis, particularly in myocytes. Here, we show that GLUT4 internalization in L6 myoblasts was inhibited in part by hypertonicity or clathrin heavy chain knockdown and in part by cholesterol depletion. Both strategies had additive effects, abolishing GLUT4 endocytosis. GLUT4 internalization was abrogated by expressing dominant-negative dynamin-2 but unaffected by inhibiting caveolar-dependent endocytosis through syntaxin-6 knockdown or caveolin mutants (which reduced lactosylceramide endocytosis). Insulin did not affect GLUT4 internalization rate or sensitivity to clathrin or cholesterol depletion. In contrast, the mitochondrial uncoupler dinitrophenol (DNP), which like insulin increases surface GLUT4, reduced GLUT4 (but not transferrin) internalization, an effect additive to that of depleting clathrin but not cholesterol. Trout GLUT4 (a natural variant of GLUT4 bearing different endocytic motifs) exogenously expressed in mammalian L6 cells internalized only through the cholesterol-dependent route that also included the non-clathrin-dependent cargo interleukin-2 receptor β, and DNP reduced internalization of both proteins. These results suggest that in muscle cells, GLUT4 internalizes simultaneously through clathrin-mediated endocytosis and a caveolae-independent but cholesterol- and dynamin-dependent route. Manipulating GLUT4 endocytosis to maintain surface GLUT4 may bypass insulin resistance.     

Loss of Myosin VI No Insert Isoform (NoI) Induces a Defect in Clathrin-mediated Endocytosis and Leads to Caveolar Endocytosis of Transferrin Receptor

Myosin VI is a motor protein that moves toward the minus end of actin filaments. It is involved in clathrin-mediated endocytosis and associates with clathrin-coated pits/vesicles at the plasma membrane. In this article the effect of the loss of myosin VI no insert isoform (NoI) on endocytosis in nonpolarized cells was examined. The absence of myosin VI in fibroblasts derived from the Snell''s waltzer mouse (myosin VI knock-out) gives rise to defective clathrin-mediated endocytosis with shallow clathrin-coated pits and a strong reduction in the internalization of clathrin-coated vesicles. To compensate for this defect in clathrin-mediated endocytosis, plasma membrane receptors such as the transferrin receptor (TfR) are internalized by a caveola-dependent pathway. Moreover the clathrin adaptor protein, AP-2, necessary for TfR internalization, follows the receptor and relocalizes in caveolae in Snell''s waltzer fibroblasts.     

Clathrin-dependent APP endocytosis and Aβ secretion are highly sensitive to the level of plasma membrane cholesterol

Several lines of evidence support a strong relationship between cholesterol and Alzheimer's disease pathogenesis. Membrane cholesterol is known to modulate amyloid precursor protein (APP) endocytosis and amyloid-β (Aβ) secretion. Here we show in a human cell line model of endocytosis (HEK293 cells) that cholesterol exerts these effects in a dose-dependent and linear manner, over a wide range of concentrations (-40% to + 40% variations of plasma membrane cholesterol induced by methyl-beta-cyclodextrin (MBCD) and MBCD-cholesterol complex respectively). We found that the gradual effect of cholesterol is inhibited by small interference RNA-mediated downregulation of clathrin. Modulation of clathrin-mediated APP endocytosis by cholesterol was further demonstrated using mutants of proteins involved in the formation of early endosomes (dynamin2, Eps15 and Rab5). Importantly we show that membrane proteins other than APP are not affected by cholesterol to the same extent. Indeed clathrin-dependent endocytosis of transferrin and cannabinoid1 receptors as well as internalization of surface proteins labelled with a biotin derivative (sulfo-NHS-SS-biotin) were not sensitive to variations of plasma membrane cholesterol from -40% to 40%. In conclusion clathrin-dependent APP endocytosis appears to be very sensitive to the levels of membrane cholesterol. These results suggest that cholesterol increase in AD could be responsible for the enhanced internalization of clathrin-, dynamin2-, Eps15- and Rab5-dependent endocytosis of APP and the ensuing overproduction of Aβ.     

Lipoplex-mediated transfection of mammalian cells occurs through the cholesterol-dependent clathrin-mediated pathway of endocytosis

Synthetic amphiphiles are widely used as a carrier system. However, to match transfection efficiencies as obtained for viral vectors, further insight is required into the properties of lipoplexes that dictate transfection efficiency, including the mechanism of delivery. Although endocytosis is often referred to as the pathway of lipoplex entry and transfection, its precise nature has been poorly defined. Here, we demonstrate that lipoplex-mediated transfection is inhibited by more than 80%, when plasma membrane cholesterol is depleted with methyl-beta-cyclodextrin. Cholesterol replenishment restores the transfection capacity. Investigation of the cellular distribution of lipoplexes after cholesterol depletion revealed an exclusive inhibition of internalization, whereas cell-association remained unaffected. These data strongly support the notion that complex internalization, rather than the direct translocation of plasmid across the plasma membrane, is a prerequisite for accomplishing effective lipoplex-mediated transfection. We demonstrate that internalized lipoplexes colocalize with transferrin in early endocytic compartments and that lipoplex internalization is inhibited in potassium-depleted cells and in cells overexpressing dominant negative Eps15 mutants. In conjunction with the notion that caveolae-mediated internalization can be excluded, we conclude that efficient lipoplex-mediated transfection requires complex internalization via the cholesterol-dependent clathrin-mediated pathway of endocytosis.     

Insulin-responsive aminopeptidase trafficking in 3T3-L1 adipocytes

The insulin-responsive aminopeptidase (IRAP/VP165/gp160) was identified originally in GLUT4-containing vesicles and shown to translocate in response to insulin, much like the glucose transporter 4 (GLUT4). This study characterizes the trafficking and kinetics of IRAP in exocytosis, endocytosis, and recycling to the membrane in 3T3-L1 adipocytes. After exposure of 3T3-L1 adipocytes to insulin, IRAP translocated to the plasma membrane as assessed by either cell fractionation, surface biotinylation, or the plasma membrane sheet assay. The rate of exocytosis closely paralleled that of GLUT4. In the continuous presence of insulin, IRAP was endocytosed with a half-time of about 3-5 min. IRAP endocytosis is inhibited by cytosol acidification, a property of clathrin-mediated endocytosis, but not by the expression of a constitutively active Akt/PKB. Arrival in an LDM fraction derived via subcellular fractionation exhibited a slower time course than disappearance from the cell surface, suggesting additional endocytic intermediates. As assayed by membrane "sheets," GLUT4 and IRAP showed similar internalization rates that are wortmannin-insensitive and occur with a half-time of roughly 5 min. IRAP remaining on the cell surface 10 min following insulin removal was both biotin- and avidin-accessible, implying the absence of thin-necked invaginations. Finally, endocytosed IRAP quickly recycled back to the plasma membrane in a wortmannin-sensitive process. These results demonstrate rapid endocytosis and recycling of IRAP in the presence of insulin and trafficking that matches GLUT4 in rate.     

The Listeria protein internalin B mimics hepatocyte growth factor-induced receptor trafficking

Increased hepatocyte growth factor receptor (HGFR) signaling correlates closely with neoplastic invasion and metastatic potential of many human cancers. Hepatocyte growth factor receptor signaling is initiated by binding the physiological ligand HGF or the internalin B (InlB) protein of Listeria monocytogenes. Subsequent degradation of endocytosed HGFR terminates receptor signaling. Previously reported discrepancies in InlB and HGF-induced HGFR signaling could reflect differences in receptor internalization and degradation in response to these distinct ligands. We report that soluble InlB and HGF are mechanistically equivalent in triggering clathrin-dependent endocytosis and lysosomal degradation of HGFR. After internalization, InlB and HGF colocalize with Rab5, EEA1 and the transferrin receptor in classical early endosomes. Hepatocyte growth factor receptor internalization was prevented by overexpression of dominant negative mutants of dynamin 1 and epidermal growth factor phosphorylation substrate 15, but not caveolin 1, the GTPase Arf6 or the cholesterol-chelating drug Nystatin. Thus, HGFR internalization is principally clathrin-mediated and is not regulated by clathrin- independent pathways. Phosphatidylinositol 3-kinase signaling and HGF-regulated tyrosine kinase substrate were not required for ligand-triggered internalization of HGFR but were essential for subsequent lysosomal degradation. Thus, soluble InlB and HGF induce HGFR endocytosis and degradation by indistinguishable mechanisms, suggesting that InlB may be exploited to regulate pathogenic HGFR signaling.     

Caveolin-1-mediated apolipoprotein A-I membrane binding sites are not required for cholesterol efflux

Caveolin-1 (Cav1), a structural protein required for the formation of invaginated membrane domains known as caveolae, has been implicated in cholesterol trafficking and homeostasis. Here we investigated the contribution of Cav1 to apolipoprotein A-I (apoA-I) cell surface binding and intracellular processing using mouse embryonic fibroblasts (MEFs) derived from wild type (WT) or Cav1-deficient (Cav1(-/-)) animals. We found that cells expressing Cav1 have 2.6-fold more apoA-I binding sites than Cav1(-/-) cells although these additional binding sites are not associated with detergent-free lipid rafts. Further, Cav1-mediated binding targets apoA-I for internalization and degradation and these processes are not correlated to cholesterol efflux. Despite lower apoA-I binding, cholesterol efflux from Cav1(-/-) MEFs is 1.7-fold higher than from WT MEFs. Stimulation of ABCA1 expression with an LXR agonist enhances cholesterol efflux from both WT and Cav1(-/-) cells without increasing apoA-I surface binding or affecting apoA-I processing. Our results indicate that there are at least two independent lipid binding sites for apoA-I; Cav1-mediated apoA-I surface binding and uptake is not linked to cholesterol efflux, indicating that membrane domains other than caveolae regulate ABCA1-mediated cholesterol efflux.     

Protein kinase B stimulates the translocation of GLUT4 but not GLUT1 or transferrin receptors in 3T3-L1 adipocytes by a pathway involving SNAP-23, synaptobrevin-2, and/or cellubrevin.

An interaction of SNAP-23 and syntaxin 4 on the plasma membrane with vesicle-associated synaptobrevin-2 and/or cellubrevin, known as SNAP (soluble N-ethyl-maleimide-sensitive factor attachment protein) receptors or SNAREs, has been proposed to provide the targeting and/or fusion apparatus for insulin-stimulated translocation of the GLUT4 isoform of glucose transporter to the plasma membrane. By microinjecting 3T3-L1 adipocytes with the Clostridium botulinum toxin B or E, which proteolyzed synaptobrevin-2/cellubrevin and SNAP-23, respectively, we investigated the role of these SNAREs in GLUT4, GLUT1, and transferrin receptor trafficking. As expected, insulin stimulated the translocation of GLUT4, GLUT1, and transferrin receptors to the plasma membrane. By contrast, a constitutively active protein kinase B (PKB-DD) only stimulated a translocation of GLUT4 and not GLUT1 or the transferrin receptor. The GLUT4 response to PKB-DD was abolished by toxins B or E, whereas the insulin-evoked translocation of GLUT4 was inhibited by approximately 65%. These toxins had no significant effect on insulin-stimulated transferrin receptor appearance at the cell surface. Thus, insulin appears to induce GLUT4 translocation via two distinct routes, only one of which involves SNAP-23 and synaptobrevin-2/cellubrevin, and can be mobilized by PKB-DD. The PKB-, SNAP-23-, and synaptobrevin-2/cellubrevin-independent GLUT4 translocation pathway may involve movement through recycling endosomes, together with GLUT1 and transferrin receptors.     

SGIP1alpha is an endocytic protein that directly interacts with phospholipids and Eps15

SGIP1 has been shown to be an endophilin-interacting protein that regulates energy balance, but its function is not fully understood. Here, we identified its splicing variant of SGIP1 and named it SGIP1alpha. SGIP1alpha bound to phosphatidylserine and phosphoinositides and deformed the plasma membrane and liposomes into narrow tubules, suggesting the involvement in vesicle formation during endocytosis. SGIP1alpha furthermore bound to Eps15, an important adaptor protein of clathrin-mediated endocytic machinery. SGIP1alpha was colocalized with Eps15 and the AP-2 complex. Upon epidermal growth factor (EGF) stimulation, SGIP1alpha was colocalized with EGF at the plasma membrane, indicating the localization of SGIP1alpha at clathrin-coated pits/vesicles. SGIP1alpha overexpression reduced transferrin and EGF endocytosis. SGIP1alpha knockdown reduced transferrin endocytosis but not EGF endocytosis; this difference may be due to the presence of redundant pathways in EGF endocytosis. These results suggest that SGIP1alpha plays an essential role in clathrin-mediated endocytosis by interacting with phospholipids and Eps15.     

Human rhinovirus type 2 is internalized by clathrin-mediated endocytosis

Using several approaches, we investigated the importance of clathrin-mediated endocytosis in the uptake of human rhinovirus serotype 2 (HRV2). By means of confocal immunofluorescence microscopy, we show that K(+) depletion strongly reduces HRV2 internalization. Viral uptake was also substantially reduced by extraction of cholesterol from the plasma membrane with methyl-beta-cyclodextrin, which can inhibit clathrin-mediated endocytosis. In accordance with these data, overexpression of dynamin K44A in HeLa cells prevented HRV2 internalization, as judged by confocal immunofluorescence microscopy, and strongly reduced infection. We also demonstrate that HRV2 bound to the surface of HeLa cells is localized in coated pits but not in caveolae. Finally, transient overexpression of the specific dominant-negative inhibitors of clathrin-mediated endocytosis, the SH3 domain of amphiphysin and the C-terminal domain of AP180, potently inhibited internalization of HRV2. Taken together, these results indicate that HRV2 uses clathrin-mediated endocytosis to infect cells.     

Identification of an adaptor-associated kinase, AAK1, as a regulator of clathrin-mediated endocytosis

The mu 2 subunit of the AP2 complex is known to be phosphorylated in vitro by a copurifying kinase, and it has been demonstrated recently that mu 2 phosphorylation is required for transferrin endocytosis (Olusanya, O., P.D. Andrews, J.R. Swedlow, and E. Smythe. 2001. Curr. Biol. 11:896-900). However, the identity of the endogenous kinase responsible for this phosphorylation is unknown. Here we identify and characterize a novel member of the Prk/Ark family of serine/threonine kinases, adaptor-associated kinase (AAK)1. We find that AAK1 copurifies with adaptor protein (AP)2 and that it directly binds the ear domain of alpha-adaptin in vivo and in vitro. In neuronal cells, AAK1 is enriched at presynaptic terminals, whereas in nonneuronal cells it colocalizes with clathrin and AP2 in clathrin-coated pits and at the leading edge of migrating cells. AAK1 specifically phosphorylates the mu subunit in vitro, and stage-specific assays for endocytosis show that mu phosphorylation by AAK1 results in a decrease in AP2-stimulated transferrin internalization. Together, these results provide strong evidence that AAK1 is the endogenous mu 2 kinase and plays a regulatory role in clathrin-mediated endocytosis. These results also lend support to the idea that clathrin-mediated endocytosis is controlled by cycles of phosphorylation/desphosphorylation.     

Cholesterol-sensitive modulation of transcytosis

Cholesterol-rich membrane domains (e.g., lipid rafts) are thought to act as molecular sorting machines, capable of coordinating the organization of signal transduction pathways within limited regions of the plasma membrane and organelles. The significance of these domains in polarized postendocytic sorting is currently not understood. We show that dimeric IgA stimulates the incorporation of its receptor into cholesterol-sensitive detergent-resistant membranes confined to the basolateral surface/basolateral endosomes. A fraction of human transferrin receptor was also found in basolateral detergent-resistant membranes. Disrupting these membrane domains by cholesterol depletion (using methyl-beta-cyclodextrin) before ligand-receptor internalization caused depolarization of traffic from endosomes, suggesting that cholesterol in basolateral lipid rafts plays a role in polarized sorting after endocytosis. In contrast, cholesterol depletion performed after ligand internalization stimulated cargo transcytosis. It also stimulated caveolin-1 phosphorylation on tyrosine 14 and the appearance of the activated protein in dimeric IgA-containing apical organelles. We propose that cholesterol depletion stimulates the coupling of transcytotic and caveolin-1 signaling pathways, consequently prompting the membranes to shuttle from endosomes to the plasma membrane. This process may represent a unique compensatory mechanism required to maintain cholesterol balance on the cell surface of polarized epithelia.     

Caveolin-1 interacts directly with dynamin-2

Caveolin is the principal component of caveolae in vivo. In addition to a structural role, it is believed to play a scaffolding function to organize and inactivate signaling molecules that are concentrated on the cytoplasmic surface of caveolar membranes. The large GTPase dynamin has been shown to mediate the scission of caveolae from the plasma membrane, although it is unclear if dynamin interacts directly with caveolin or via accessory proteins. Therefore, the goal of this study was to test whether dynamin associates with caveolae via a direct binding to the caveolin 1 (Cav1) protein. Immunoelectron microscopy of lung endothelium or a cultured hepatocyte cell line stained with antibodies for Dyn2 and Cav1 shows that these proteins co-localize to caveolae. To further define this interaction biochemically, in vitro experiments were performed using glutathione-S-transferase (GST)-Dyn2 and GST-Cav1 fusion proteins, which demonstrated a direct interaction between these proteins. This interaction appears to be mediated by the proline-arginine-rich domain (PRD) of Dyn2, as a GST-PRD fragment binds Cav1 while GST-Dyn2DeltaPRD does not. Further, in vitro binding studies using two Dyn2 spliced forms and Cav1 peptides immobilized on paper identify specific domains of Cav1 that bind Dyn2. Interestingly, these Cav1-binding domains differ markedly between two spliced variant forms of Dyn2. In support of these distinctive physical interactions, we find that the different Dyn2 forms, when expressed as GTPase-defective mutants, exert markedly different inhibitory effects on caveolae internalization, as assayed by cholera toxin uptake. These studies provide the first evidence for a direct interaction between dynamin and the caveolin coat, and demonstrate a selectivity of one Dyn2 form toward the caveolae-mediated endocytosis.     

EHD2 and the novel EH domain binding protein EHBP1 couple endocytosis to the actin cytoskeleton

Here we identified two novel proteins denoted EH domain protein 2 (EHD2) and EHD2-binding protein 1 (EHBP1) that link clathrin-mediated endocytosis to the actin cytoskeleton. EHD2 contains an N-terminal P-loop and a C-terminal EH domain that interacts with NPF repeats in EHBP1. Disruption of EHD2 or EHBP1 function by small interfering RNA-mediated gene silencing inhibits endocytosis of transferrin into EEA1-positive endosomes as well as GLUT4 endocytosis into cultured adipocytes. EHD2 localizes with cortical actin filaments, whereas EHBP1 contains a putative actin-binding calponin homology domain. High expression of EHD2 or EHBP1 in intact cells mediates extensive actin reorganization. Thus EHD2 appears to connect endocytosis to the actin cytoskeleton through interactions of its N-terminal domain with membranes and its C-terminal EH domain with the novel EHBP1 protein.     

The amino terminus of GLUT4 functions as an internalization motif but not an intracellular retention signal when substituted for the transferrin receptor cytoplasmic domain

Previous studies have demonstrated that the amino-terminal cytoplasmic domain of GLUT4 contains a phenylalanine-based targeting motif that determines its steady state distribution between the surface and the interior of cells (Piper, R. C., C. Tai, P. Kuleza, S. Pang, D. Warnock, J. Baenziger, J. W. Slot, H. J. Geuze, C. Puri, and D. E. James. 1993. J. Cell Biol. 121:1221). To directly measure the effect that the GLUT4 amino terminus has on internalization and subsequent recycling back to the cell surface, we constructed chimeras in which this sequence was substituted for the amino-terminal cytoplasmic domain of the human transferrin receptor. The chimeras were stably transfected into Chinese hamster ovary cells and their endocytic behavior characterized. The GLUT4-transferrin receptor chimera was recycled back to the cell surface with a rate similar to the transferrin receptor, indicating that the GLUT4 sequence was not promoting intracellular retention of the chimera. The GLUT4-transferrin receptor chimera was internalized at half the rate of the transferrin receptor. Substitution of an alanine for phenylalanine at position 5 slowed internalization of the chimera by twofold, to a level characteristic of bulk membrane internalization. However, substitution of a tyrosine increased the rate of internalization to the level of the transferrin receptor. Neither of these substitutions significantly altered the rate at which the chimeras were recycled back to the cell surface. These results demonstrate that the major function of the GLUT4 amino-terminal domain is to promote the effective internalization of the protein from the cell surface, via a functional phenylalanine-based internalization motif, rather than retention of the transporter within intracellular structures.     

Analysis of clathrin-mediated endocytosis of epidermal growth factor receptor by RNA interference

To identify proteins that participate in clathrin-mediated endocytosis of the epidermal growth factor receptor (EGFR), 13 endocytic proteins were depleted in HeLa cells using highly efficient small interfering RNAs that were designed using a novel selection algorithm. The effects of small interfering RNAs on the ligand-induced endocytosis of EGFR were compared with those effects on the constitutive internalization of the transferrin receptor. The knock-downs of clathrin heavy chain and dynamin produced maximal inhibitory effects on the internalization of both receptors. Depletion of alpha, beta2, or micro2 subunits of AP-2 reduced EGF and transferrin internalization rates by 40-60%. Down-regulation of several accessory proteins individually had no effect on endocytosis but caused significant inhibition of EGF and transferrin endocytosis when the homologous proteins were depleted simultaneously. Surprisingly, knockdown of clathrin-assembly lymphoid myeloid leukemia protein, CALM, did not influence transferrin endocytosis but considerably affected EGFR internalization. Thus, CALM is the second protein besides Grb2 that appears to play a specific role in EGFR endocytosis. This study demonstrates that the efficient gene silencing by rationally designed small interfering RNA can be used as an approach to functionally analyze the entire cellular machineries, such as the clathrin-coated pits and vesicles.     

GLUT4 distribution between the plasma membrane and the intracellular compartments is maintained by an insulin-modulated bipartite dynamic mechanism

The GLUT4 glucose transporter is predominantly retained inside basal fat and muscle cells, and it is rapidly recruited to the plasma membrane with insulin stimulation. There is controversy regarding the mechanism of basal GLUT4 retention. One model is that GLUT4 retention is dynamic, based on slow exocytosis and rapid internalization of the entire pool of GLUT4 (Karylowski, O., Zeigerer, A., Cohen, A., and McGraw, T. E. (2004) Mol. Biol. Cell 15, 870-882). In this model, insulin increases GLUT4 in the plasma membrane by modulating GLUT4 exocytosis and endocytosis. The second model is that GLUT4 retention is static, with approximately 90% of GLUT4 stored in compartments that are not in equilibrium with the cell surface in basal conditions (Govers, R., Coster, A. C., and James, D. E. (2004) Mol. Cell Biol. 24, 6456-6466). In this model, insulin increases GLUT4 in the plasma membrane by releasing it from the static storage compartment. Here we show that under all experimental conditions examined, basal GLUT4 retention is by a bipartite dynamic mechanism involving slow efflux and rapid internalization. To establish that the dynamic model developed in studies of the extreme conditions of >100 nm insulin and no insulin also describes GLUT4 behavior at more physiological insulin concentrations, we characterized GLUT4 trafficking in 0.5 nm insulin. This submaximal insulin concentration promotes an intermediate effect on both GLUT4 exocytosis and endocytosis, resulting in an intermediate degree of redistribution to the plasma membrane. These data establish that changes in the steady-state surface/total distributions of GLUT4 are the result of gradated, insulin-induced changes in GLUT4 exocytosis and endocytosis rates.     

Caveolin-1 is a negative regulator of caveolae-mediated endocytosis to the endoplasmic reticulum.

Caveolae are flask-shaped invaginations at the plasma membrane that constitute a subclass of detergent-resistant membrane domains enriched in cholesterol and sphingolipids and that express caveolin, a caveolar coat protein. Autocrine motility factor receptor (AMF-R) is stably localized to caveolae, and the cholesterol extracting reagent, methyl-beta-cyclodextrin, inhibits its internalization to the endoplasmic reticulum implicating caveolae in this distinct receptor-mediated endocytic pathway. Curiously, the rate of methyl-beta-cyclodextrin-sensitive endocytosis of AMF-R to the endoplasmic reticulum is increased in ras- and abl-transformed NIH-3T3 cells that express significantly reduced levels of caveolin and few caveolae. Overexpression of the dynamin K44A dominant negative mutant via an adenovirus expression system induces caveolar invaginations sensitive to methyl-beta-cyclodextrin extraction in the transformed cells without increasing caveolin expression. Dynamin K44A expression further inhibits AMF-R-mediated endocytosis to the endoplasmic reticulum in untransformed and transformed NIH-3T3 cells. Adenoviral expression of caveolin-1 also induces caveolae in the transformed NIH-3T3 cells and reduces AMF-R-mediated endocytosis to the endoplasmic reticulum to levels observed in untransformed NIH-3T3 cells. Cholesterol-rich detergent-resistant membrane domains or glycolipid rafts therefore invaginate independently of caveolin-1 expression to form endocytosis-competent caveolar vesicles via rapid dynamin-dependent detachment from the plasma membrane. Caveolin-1 stabilizes the plasma membrane association of caveolae and thereby acts as a negative regulator of the caveolae-mediated endocytosis of AMF-R to the endoplasmic reticulum.     

Endocytosis of oxidized low density lipoprotein through scavenger receptor CD36 utilizes a lipid raft pathway that does not require caveolin-1

The scavenger receptor CD36 binds a diverse array of ligands, including thrombospondin-1, oxidized low density lipoprotein (OxLDL), fatty acids, anionic phospholipids, and apoptotic cells. CD36 has been reported to be present in lipid rafts/caveolae, but little is known about the membrane trafficking of this protein at baseline or following ligand binding. Here, we determined that expression of CD36 in Chinese hamster ovary (CHO) cells and endogenous expression of CD36 in C32 cells led to a homogeneous distribution of the protein on the plasma membrane, as judged by confocal fluorescence microscopy. This homogeneous pattern was observed both by anti-CD36 antibody staining and by live cell imaging of CHO cells expressing a chimeric CD36-green fluorescent protein construct. In contrast, caveolin-1 displayed its usual punctate surface distribution. Correspondingly, dual labeling of CD36 and caveolin-1 showed essentially no overlap, neither by immunofluorescence light microscopy nor by immunogold electron microscopy. Furthermore, isolation of lipid rafts by sucrose gradient ultracentrifugation of cold Triton X-100 cell lysates yielded both CD36 and caveolin-1, but immunoprecipitates of caveolin-1 did not contain CD36. Binding of Ox-LDL led to internalization of CD36 and OxLDL into endosomal structures that did not contain caveolin-1 or transferrin but that co-internalized the glycosyl-phosphatidylinositol-anchored protein decay accelerating factor, a lipid raft protein. Furthermore, expression of CD36 in the caveolin-1-negative KB cell line is sufficient for OxLDL-induced internalization of CD36, indicating that caveolin-1 is not required for this endocytic process. Taken together, these data demonstrate that at steady state, CD36 is localized in lipid rafts but not in caveolae, and that binding of OxLDL to CD36 leads to endocytosis through a lipid raft pathway that is distinct from the clathrin-mediated or caveolin internalization pathways.     

Ubiquitylation of leptin receptor OB-Ra regulates its clathrin-mediated endocytosis

Leptin receptors are constitutively endocytosed in a ligand-independent manner. To study their endocytosis, leptin receptors OB-Ra and OB-Rb were expressed in HeLa cells. Both receptor isoforms were ubiquitylated, internalized by clathrin-mediated endocytosis and transported to Hrs-positive endosomes after their internalization. Proteasome inhibitors inhibited OB-Ra but not OB-Rb internalization from the cell surface. OB-Ra ubiquitylation occurred on lysine residues K877 and K889 in the cytoplasmic tail, the mutation of which abolished OB-Ra internalization. Fusion of an ubiquitin molecule at the C-terminus of an OB-Ra construct defective both in ubiquitylation and endocytosis restored clathrin-dependent endocytosis of the receptor. The internalization of this constitutively mono-ubiquitylated construct was no longer sensitive to proteasome inhibitors, which inhibited OB-Ra endocytosis by blocking its ubiquitylation. Fusion of an ubiquitin molecule to a transferrin receptor deleted from its own endocytosis motif restored clathrin-mediated endocytosis. We propose that mono-ubiquitin conjugates act as internalization motifs for clathrin-dependent endocytosis of leptin receptor OB-Ra.