Sequestration of epidermal growth factor receptors in non-caveolar lipid rafts inhibits ligand binding

Cholesterol depletion has been shown to increase mitogen-activated protein kinase activation in response to stimulation with epidermal growth factor (EGF) (Furuchi, T., and Anderson, R. G. W. (1998) J. Biol. Chem. 273, 21099-21104). However, the underlying mechanisms are unknown. We show that cholesterol depletion increases EGF binding, whereas cholesterol loading lowers EGF binding. Based on binding analyses, we demonstrate that the observed changes in EGF binding are caused by alterations in the number of EGF receptors available for ligand binding, whereas the affinity of the receptor for EGF remains unaltered. We also show by immunofluorescence that in unstimulated cells the EGF receptor is localized in non-caveolar lipid rafts containing the ganglioside GM1 and that patching of these rafts by cholera toxin B-chain causes co-patching of EGF receptors. Experiments with solubilization in different detergents at 4 degrees C show that the association of the EGF receptor with these rafts is sensitive to Triton X-100 extraction but insensitive to extraction with another non-ionic detergent, Brij 58. Furthermore, experiments with cholesterol-depleted cells show that the association is cholesterol-dependent. We propose that non-caveolar lipid rafts function as negative regulators of EGF receptor signaling by sequestering a fraction of the EGF receptors in a state inaccessible for ligand binding.     

Cholesterol depletion results in site-specific increases in epidermal growth factor receptor phosphorylation due to membrane level effects. Studies with cholesterol enantiomers

In A431 cells, depletion of cholesterol with methyl-beta-cyclodextrin induced an increase in both basal and epidermal growth factor (EGF)-stimulated EGF receptor phosphorylation. This increase in phosphorylation was site-specific, with significant increases occurring at Tyr845, Tyr992, and Tyr1173, but only minor changes at Tyr1045 and Tyr1068. The elevated level of receptor phosphorylation was associated with an increase in the intrinsic kinase activity of the EGF receptor kinase, possibly as a result of the cyclodextrin-induced enhancement of the phosphorylation of Tyr845, a site in the kinase activation loop known to be phosphorylated by pp60src. Cholesterol and its enantiomer (ent-cholesterol) were used to investigate the molecular basis for the modulation of EGF receptor function by cholesterol. Natural cholesterol (nat-cholesterol) was oxidized substantially more rapidly than ent-cholesterol by cholesterol oxidase, a protein that contains a specific binding site for the sterol. By contrast, the ability of nat- and ent-cholesterol to interact with sphingomyelins and phosphatidylcholine and to induce lipid condensation in a monolayer system was the same. These data suggest that, whereas cholesterol-protein interactions may be sensitive to the absolute configuration of the sterol, sterol-lipid interactions are not. nat- and ent-cholesterol were tested for their ability to physically reconstitute lipid rafts following depletion of cholesterol. nat- and ent-cholesterol reversed to the same extent the enhanced phosphorylation of the EGF receptor that occurred following removal of cholesterol. Furthermore, the enantiomers showed similar abilities to reconstitute lipid rafts in cyclodextrin-treated cells. These data suggest that cholesterol most likely affects EGF receptor function because of its physical effects on membrane properties, not through direct enantioselective interactions with the receptor.     

Cellular functions of cholesterol probed with optical biosensors

Cholesterol is an essential constituent of cell membranes and the regulation of cholesterol concentration is critical for cell functions including signaling. In this paper, we applied resonant waveguide grating (RWG) biosensor to study the cellular functions of cholesterol through real time monitoring the dynamic mass redistribution (DMR) mediated by cholesterol depletion with methyl-beta-cyclodextrin (mbetaCD). In A431 cells, depletion of cholesterol by mbetaCD led to a DMR signature that was similar, but not identical to that induced by epidermal growth factor (EGF). To elucidate the cellular mechanisms of the DMR signal mediated by cholesterol depletion, a panel of modulators that specifically modulate the activities of various cellular targets were used to pretreat the cells. Results showed that the DMR signals triggered by cholesterol depletion are primarily linked to the transactivation of EGF receptor. Multiple signaling pathways including Ras/mitogenic activated protein (MAP) kinase, protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) acted synergically in the cell response, whereas the activation of protein kinase A (PKA) pathway was found to antagonize the cell response.     

Constitutive localization of the gonadotropin-releasing hormone (GnRH) receptor to low density membrane microdomains is necessary for GnRH signaling to ERK

Specialized membrane microdomains known as lipid rafts are thought to contribute to G-protein coupled receptor (GPCR) signaling by organizing receptors and their cognate signaling molecules into discrete membrane domains. To determine if the GnRHR, an unusual member of the GPCR superfamily, partitions into lipid rafts, homogenates of alpha T3-1 cells expressing endogenous GnRHR or Chinese hamster ovary cells expressing an epitope-tagged GnRHR were fractionated through a sucrose gradient. We found the GnRHR and c-raf kinase constitutively localized to low density fractions independent of hormone treatment. Partitioning of c-raf kinase into lipid rafts was also observed in whole mouse pituitary glands. Consistent with GnRH induced phosphorylation and activation of c-raf kinase, GnRH treatment led to a decrease in the apparent electrophoretic mobility of c-raf kinase that partitioned into lipid rafts compared with unstimulated cells. Cholesterol depletion of alpha T3-1 cells using methyl-beta-cyclodextrin disrupted GnRHR but not c-raf kinase association with rafts and shifted the receptor into higher density fractions. Cholesterol depletion also significantly attenuated GnRH but not phorbol ester-mediated activation of extracellular signal-related kinase (ERK) and c-fos gene induction. Raft localization and GnRHR signaling to ERK and c-Fos were rescued upon repletion of membrane cholesterol. Thus, the organization of the GnRHR into low density membrane microdomains appears critical in mediating GnRH induced intracellular signaling.     

Sensitization of epidermal growth factor-induced signaling by bradykinin is mediated by c-Src. Implications for a role of lipid microdomains

Communication between receptor tyrosine kinase (RTK)- and G protein-coupled receptor (GPCR)-mediated signaling systems has received increasing attention in recent years. Here, we report that activation of G protein-coupled bradykinin B2 receptor induces an up-regulation of cellular responses mediated by epidermal growth factor receptor (EGFR) and provide essential mechanistic characteristics of this sensitization process. EGF, which failed to evoke detectable amount of calcium increase and neurotransmitter release when administrated alone in primary cultures of rat adrenal chromaffin cells and PC12 cells, became capable of inducing these responses specifically after bradykinin pretreatment. Both EGFR and non-receptor tyrosine kinase p60Src, whose kinase activities were required in the sensitization, were found to be enriched in cholesterol-rich lipid rafts. Bradykinin caused activation of p60Src and Src-dependent phosphorylation of the EGFR on Tyr-845 in lipid rafts, as well as recruitment of phospholipase C (PLC) gamma1 to the rafts. Depletion of cholesterol by methyl-beta-cyclodextrin disrupted the raft localization of EGFR and Src, as well as bradykinin-induced translocation of PLCgamma1. Furthermore, sensitization, which was impaired by cholesterol depletion, was restored by repletion of cholesterol. Therefore, we suggest that lipid rafts are essential participants in the regulation of receptor-mediated signal transduction and cross-talk via organizing signaling complexes in membrane microdomains.     

Heparin suppresses lipid raft-mediated signaling and ligand-independent EGF receptor activation

Heparin is well known to suppress vascular smooth muscle cell (VSMC) proliferation, and attempts to exploit this therapeutically have led to recognition of multiple pathways for heparin's anti-mitogenic actions. At low concentrations (ca. 1 microg.ml(-1)), these suppressive effects may reflect physiological activities of endogenous heparan sulfates, and appear to be rapid responses to extracellular or cell surface-associated heparin. Because heparin has been shown to influence expression of caveolin proteins, and caveolae/lipid rafts are critical structures modulating cell signaling, we examined the effect of heparin on signaling involving cholesterol-rich membrane microdomains. The VSMC line PAC-1 activates the MAP kinase Erk in response to the cholesterol-sequestering agents methyl-beta-cyclodextrin and nystatin. This follows a temporal sequence that involves Ras-GTP activation of MEK, and is independent of PKC, Src, and PI3 kinase. However, ligand-independent phosphorylation of the EGF receptor (EGFR) by removal of cholesterol precedes Ras activation, and the EGFR kinase inhibitor AG1478 blocks Erk phosphorylation, supporting occurrence of the signaling sequence EGFR-Ras-MEK-Erk. Phosphorylation of EGFR occurs predominantly in caveolin-rich microdomains as identified by Western blotting of fractions from density gradient centrifugation of membranes prepared under detergent-free conditions. In these situations, heparin inhibits phosphorylation of EGFR on the Src-dependent site Tyr(845), but not the autophosphorylation of Tyr(1173), and decreases Ras activation and Erk phosphorylation. We conclude that heparin can suppress Erk signaling in VSMC with effects on site-specific phosphorylation of EGFR localized in caveolin-enriched lipid rafts.     

Agonist-dependent Signaling by Group I Metabotropic Glutamate Receptors Is Regulated by Association with Lipid Domains

Group I metabotropic glutamate receptors (mGluRs), mGluR1 and mGluR5, play critical functions in forms of activity-dependent synaptic plasticity and synapse remodeling in physiological and pathological states. Importantly, in animal models of fragile X syndrome, group I mGluR activity is abnormally enhanced, a dysfunction that may partly underlie cognitive deficits in the condition. Lipid rafts are cholesterol- and sphingolipid-enriched membrane domains that are thought to form transient signaling platforms for ligand-activated receptors. Many G protein-coupled receptors, including group I mGluRs, are present in lipid rafts, but the mechanisms underlying recruitment to these membrane domains remain incompletely understood. Here, we show that mGluR1 recruitment to lipid rafts is enhanced by agonist binding and is supported at least in part by an intact cholesterol recognition/interaction amino acid consensus (CRAC) motif in the receptor. Substitutions of critical residues in the motif reduce mGluR1 association with lipid rafts and agonist-induced, mGluR1-dependent activation of extracellular-signal-activated kinase1/2 MAP kinase (ERK-MAPK). We find that alteration of membrane cholesterol content or perturbation of lipid rafts regulates agonist-dependent activation of ERK-MAPK by group I mGluRs, suggesting a potential function for cholesterol as a positive allosteric modulator of receptor function(s). Together, these findings suggest that drugs that alter membrane cholesterol levels or directed to the receptor-cholesterol interface could be employed to modulate abnormal group I mGluR activity in neuropsychiatric conditions, including fragile X syndrome.     

Cholesterol depletion inhibits epidermal growth factor receptor transactivation by angiotensin II in vascular smooth muscle cells: role of cholesterol-rich microdomains and focal adhesions in angiotensin II signaling

Angiotensin II (Ang II) induces transactivation of the epidermal growth factor (EGF) receptor (EGF-R), which serves as a scaffold for various signaling molecules in vascular smooth muscle cells (VSMCs). Cholesterol and sphingomyelin-enriched lipid rafts are plasma membrane microdomains that concentrate various signaling molecules. Caveolae are specialized lipid rafts that are organized by the cholesterol-binding protein, caveolin, and have been shown to be associated with EGF-Rs. Angiotensin II stimulation promotes a rapid movement of AT(1) receptors to caveolae; however, their functional role in angiotensin II signaling has not been elucidated. Here we show that cholesterol depletion by beta-cyclodextrin disrupts caveolae structure and concomitantly inhibits tyrosine phosphorylation of the EGF-R and subsequent activation of protein kinase B (PKB)/Akt induced by angiotensin II. Similar inhibitory effects were obtained with other cholesterol-binding agents, filipin and nystatin. In contrast, EGF-R autophosphorylation and activation of Akt/PKB in response to EGF are not affected by cholesterol depletion. The early Ang II-induced upstream signaling events responsible for transactivation of the EGF-R, such as the intracellular Ca(2+) increase and c-Src activation, also remain intact. The EGF-R initially binds caveolin, but these two proteins rapidly dissociate following angiotensin II stimulation during the time when EGF-R transactivation is observed. The activated EGF-R is localized in focal adhesions together with tyrosine-phosphorylated caveolin. These findings suggest that 1) a scaffolding role of caveolin is essential for EGF-R transactivation by angiotensin II and 2) cholesterol-rich microdomains as well as focal adhesions are important signal-organizing compartments required for the spatial and temporal organization of angiotensin II signaling in VSMCs.     

Cholesterol depletion induces ANTXR2-dependent activation of MMP-2 via ERK1/2 phosphorylation in neuroglioma U251 cell

Cholesterol is a critical component of lipid rafts implicated in regulating multiple signal transduction. The anthrax toxin receptor 2 (ANTXR2) is a type I membrane protein acting as the second receptor for the anthrax toxin. In this study, we first investigated the association between cholesterol and ANTXR2. We provided the evidence that cholesterol depletion by methyl-beta-cyclodextrin (MβCD) promoted ANTXR2 expression in U251 neuroglioma cell, which was reversed by cholesterol supplement. MβCD-induced ANTXR2 up-regulation contributed to ERK1/2 phosphorylation, which was responsible for MT1-MMP and MMP-2 activation. Our data suggested that cellular cholesterol regulated ANTXR2-dependent activation of MMP-2 via ERK1/2 phosphorylation in neuroglioma U251 cell.     

The glycoprotein Ib-IX-V complex mediates localization of factor XI to lipid rafts on the platelet membrane

Factor XI binds to activated platelets where it is efficiently activated by thrombin. The factor XI receptor is the platelet membrane glycoprotein (GP) Ib-IX-V complex (Baglia, F. A., Badellino, K. O., Li, C. Q., Lopez, J. A., and Walsh, P. N. (2002) J. Biol. Chem. 277, 1662-1668), a significant fraction of which exists within lipid rafts on stimulated platelets (Shrimpton, C. N., Borthakur, G., Larrucea, S., Cruz, M. A., Dong, J. F., and Lopez, J. A. (2002) J. Exp. Med. 196, 1057-1066). Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids implicated in localizing membrane ligands and in cellular signaling. We now show that factor XI was localized to lipid rafts in activated platelets ( approximately 8% of total bound) but not in resting platelets. Optimal binding of factor XI to membrane rafts required prothrombin (and Ca2+) or high molecular weight kininogen (and Zn2+), which are required for factor XI binding to platelets. An antibody to GPIb (SZ-2) that disrupts factor XI binding to the GPIb-IX-V complex also disrupted factor XI-raft association. The isolated recombinant Apple 3 domain of factor XI, which mediates factor XI binding to platelets, also completely displaces factor XI from membrane rafts. To investigate the physiological relevance of the factor XI-raft association, the structural integrity of lipid rafts was disrupted by cholesterol depletion utilizing methyl-beta-cyclodextrin. Cholesterol depletion completely prevented FXI binding to lipid rafts, and initial rates of factor XI activation by thrombin on activated platelets were inhibited >85%. We conclude that factor XI is localized to GPIb in membrane rafts and that this association is important for promoting the activation of factor XI by thrombin on the platelet surface.     

Plasma membrane and lysosomal localization of CB1 cannabinoid receptor are dependent on lipid rafts and regulated by anandamide in human breast cancer cells

In this report we show, by confocal analysis of indirect immunofluorescence, that the type-1 cannabinoid receptor (CB1R), which belongs to the family of G-protein-coupled receptors, is expressed on the plasma membrane in human breast cancer MDA-MB-231 cells. However, a substantial proportion of the receptor is present in lysosomes. We found that CB1R is associated with cholesterol- and sphyngolipid-enriched membrane domains (rafts). Cholesterol depletion by methyl-beta-cyclodextrin (MCD) treatment strongly reduces the flotation of the protein on the raft-fractions (DRM) of sucrose density gradients suggesting that CB1 raft-association is cholesterol dependent. Interestingly binding of the agonist, anandamide (AEA) also impairs DRM-association of the receptor suggesting that the membrane distribution of the receptor is dependent on rafts and is possibly regulated by the agonist binding. Indeed MCD completely blocked the clustering of CB1R at the plasma membrane. On the contrary the lysosomal localization of CB1R was impaired by this treatment only after AEA binding.     

Lipid rafts regulate cellular CD40 receptor localization in vascular endothelial cells

Cholesterol enriched lipid rafts are considered to function as platforms involved in the regulation of membrane receptor signaling complex through the clustering of signaling molecules. In this study, we tested whether these specialized membrane microdomains affect CD40 localization in vitro and in vivo. Here, we provide evidence that upon CD40 ligand stimulation, endogenous and exogenous CD40 receptor is rapidly mobilized into lipid rafts compared with unstimulated HAECs. Efficient binding between CD40L and CD40 receptor also increases amounts of CD40 protein levels in lipid rafts. Deficiency of intracellular conserved C terminus of the CD40 cytoplasmic tail impairs CD40 partitioning in raft. Raft disorganization after methyl-beta-cyclodextrin treatment diminishes CD40 localization into rafts. In vivo studies show that elevation of circulating cholesterol in high-cholesterol fed rabbits increases the cholesterol content and CD40 receptor localization in lipid rafts. These findings identify a physiological role for membrane lipid rafts as a critical regulator of CD40-mediated signal transduction and raise the possibility that certain pathologic conditions may be treated by altering CD40 signaling with drugs affecting its raft localization.     

Lipid raft localization of cell surface E-selectin is required for ligation-induced activation of phospholipase C gamma

E-selectin, an endothelial cell surface adhesion receptor for leukocytes, also acts as a signaling receptor. Upon multivalent ligation, E-selectin transduces outside-in signals into the endothelium leading to changes in intracellular Ca(2+) concentration and activation of the mitogen-activated protein kinase signaling pathway. In addition, following leukocyte engagement, E-selectin associates via its cytoplasmic domain with components of the actin cytoskeleton and undergoes alterations in phosphorylation state that result in changes in gene expression. In this study, we show that E-selectin is localized in cholesterol-rich lipid rafts at the cell surface, and that upon ligation E-selectin clusters and redistributes in the plasma membrane colocalizing with a fraction of caveolin-1-containing rafts. In addition, we demonstrate that leukocyte adhesion via E-selectin results in association with and activation of phospholipase Cgamma (PLCgamma). Moreover, we show that disruption of lipid rafts with the cholesterol-depleting drug methyl-beta-cyclodextrin disrupts the raft localization of E-selectin as well as the ligation-induced association of E-selectin with PLCgamma, and subsequent tyrosine phosphorylation of PLCgamma. In contrast, cholesterol depletion has no effect on E-selectin-dependent mitogen-activated protein kinase activation. Thus, these findings demonstrate that the presence of E-selectin in lipid rafts is necessary for its association with, and activation of, PLCgamma, and suggest that this subcellular localization of E-selectin is related to its signaling function(s) during leukocyte-endothelial interactions.     

Lipid Raft Association and Cholesterol Sensitivity of P2X1-4 Receptors for ATP: CHIMERAS AND POINT MUTANTS IDENTIFY INTRACELLULAR AMINO-TERMINAL RESIDUES INVOLVED IN LIPID REGULATION OF P2X1 RECEPTORS*

Cholesterol-rich lipid rafts act as signaling microdomains and can regulate receptor function. We have shown in HEK293 cells recombinant P2X1-4 receptors (ATP-gated ion channels) are expressed in lipid rafts. Localization to flotillin-rich lipid rafts was reduced by the detergent Triton X-100. This sensitivity to Triton X-100 was concentration- and subunit-dependent, demonstrating differential association of P2X1-4 receptors with lipid rafts. The importance of raft association to ATP-evoked P2X receptor responses was determined in patch clamp studies. The cholesterol-depleting agents methyl-β-cyclodextrin or filipin disrupt lipid rafts and reduced P2X1 receptor currents by >90%. In contrast, ATP-evoked P2X2-4 receptor currents were unaffected by lipid raft disruption. To determine the molecular basis of cholesterol sensitivity, we generated chimeric receptors replacing portions of the cholesterol-sensitive P2X1 receptor with the corresponding region from the insensitive P2X2 receptor. These chimeras identified the importance of the intracellular amino-terminal region between the conserved protein kinase C site and the first transmembrane segment for the sensitivity to cholesterol depletion. Mutation of any of the variant residues between P2X1 and P2X2 receptors in this region in the P2X1 receptor (residues 20–23 and 27–29) to cysteine removed cholesterol sensitivity. Cholesterol depletion did not change the ATP sensitivity or cell surface expression of P2X1 receptors. This suggests that cholesterol is normally needed to facilitate the opening/gating of ATP-bound P2X1 receptor channels, and mutations in the pre-first transmembrane segment region remove this requirement.     

Lipid raft disruption triggers protein kinase C and Src-dependent protein kinase D activation and Kidins220 phosphorylation in neuronal cells

Kidins220 (kinase D-interacting substrate of 220 kDa) is a novel neurospecific protein recently cloned as the first substrate for the Ser/Thr kinase protein kinase D (PKD). Herein we report that Kidins220 is constitutively associated to lipid rafts in PC12 cells, rat primary cortical neurons, and brain synaptosomes. Immunocytochemistry and confocal microscopy together with sucrose gradient fractionation show co-localization of Kidins220 and lipid raft-associated proteins. In addition, cholesterol depletion of cell membranes with methyl-beta-cyclodextrin dramatically alters Kidins220 localization and detergent solubility. By studying the putative involvement of lipid rafts in PKD activation and signaling we have found that active PKD partitions in lipid raft fractions after sucrose gradient centrifugation and that green fluorescent protein-PKD translocates to lipid raft microdomains at the plasma membrane after phorbol ester treatment. Strikingly, lipid rafts disruption by methyl-beta-cyclodextrin delays green fluorescent protein-PKD translocation, as determined by live cell confocal microscopy, and activates PKD, increasing Kidins220 phosphorylation on Ser(919) by a mechanism involving PKCepsilon and the small soluble tyrosine kinase Src. Collectively, these results reveal the importance of lipid rafts on PKD activation, translocation, and downstream signaling to its substrate Kidins220.     

The NK1 receptor localizes to the plasma membrane microdomains, and its activation is dependent on lipid raft integrity

The spatial targeting of receptors to discrete domains within the plasma membrane allows their preferential coupling to specific effectors, which is essential for rapid and accurate discrimination of signals. Efficiency of signaling is further increased by protein and lipid segregation within the plasma membrane. We have previously demonstrated the importance of raft-mediated signaling in the regulation of smooth and skeletal muscle cell contraction. Since G protein-coupled receptors (GPCRs) are key components in the regulation of smooth muscle contraction-relaxation cycles, it is important to determine whether GPCR signaling is mediated by lipid rafts and raft-associated molecules. Neurokinin 1 receptor (NK1R) is expressed in central and peripheral nervous system as well as in endothelial and smooth muscle cells and involved in mediation of pain, inflammation, exocrine secretion, and smooth muscle contraction. The NK1 receptor was transiently expressed in HEK293 and HepG2 cell lines and its localization in membrane microdomains investigated using biochemical methods and immunofluorescent labeling. We show that the NK1 receptor, similar to the earlier described beta(2)-adrenergic receptor and G proteins, localizes to lipid rafts and caveolae. Protein kinase C (PKC) is one of the downstream effectors of the NK1 activation. Its active form translocates from the cytoplasm to the plasma membrane. Upon stimulation of the NK1 receptor with Substance P, the activated PKC relocated to lipid rafts. Using cholesterol extraction and replenishment assays we show that activation of NK1 receptor is dependent on the microarchitecture of the plasma membrane: NK1R-mediated signaling was abolished after cholesterol depletion of the receptor-expressing cells with methyl-beta-cyclodextrin. Our results demonstrate that reorganization of the plasma membrane has an effect on the activation of the raft-associated NK1R and the down-stream events such as recruitment of protein kinases.     

Role of lipid rafts in E-cadherin-- and HGF-R/Met--mediated entry of Listeria monocytogenes into host cells

Listeria monocytogenes uptake by nonphagocytic cells is promoted by the bacterial invasion proteins internalin and InlB, which bind to their host receptors E-cadherin and hepatocyte growth factor receptor (HGF-R)/Met, respectively. Here, we present evidence that plasma membrane organization in lipid domains is critical for Listeria uptake. Cholesterol depletion by methyl-beta-cyclodextrin reversibly inhibited Listeria entry. Lipid raft markers, such as glycosylphosphatidylinositol-linked proteins, a myristoylated and palmitoylated peptide and the ganglioside GM1 were recruited at the bacterial entry site. We analyzed which molecular events require membrane cholesterol and found that the presence of E-cadherin in lipid domains was necessary for initial interaction with internalin to promote bacterial entry. In contrast, the initial interaction of InlB with HGF-R did not require membrane cholesterol, whereas downstream signaling leading to F-actin polymerization was cholesterol dependent. Our work, in addition to documenting for the first time the role of lipid rafts in Listeria entry, provides the first evidence that E-cadherin and HGF-R require lipid domain integrity for their full activity.     

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.     

Limited cholesterol depletion causes aggregation of plasma membrane lipid rafts inducing T cell activation

Acute cholesterol depletion is generally associated with decreased or abolished T cell signalling but it can also cause T cell activation. This anomaly has been addressed in Jurkat T cells using progressive cholesterol depletion with methyl-beta-cyclodextrin (MBCD). At depletion levels higher than 50% there is substantial cell death, which explains reports of signalling inhibition. At 10–20% depletion levels, tyrosine phosphorylation is increased, ERK is activated and there is a small increase in cytoplasmic Ca²⁺. Peripheral actin polymerisation is also triggered by limited cholesterol depletion. Strikingly, the lipid raft marker GM1 aggregates upon cholesterol depletion and these aggregated domains concentrate the signalling proteins Lck and LAT, whereas the opposite is true for the non lipid raft marker the transferrin receptor. Using PP2, an inhibitor of Src family kinase activation, it is demonstrated that the lipid raft aggregation occurs independently of and thus upstream of the signalling response. Upon cholesterol depletion there is an increase in overall plasma membrane order, indicative of more ordered domains forming at the expense of disordered domains. That cholesterol depletion and not unspecific effects of MBCD was behind the reported results was confirmed by performing all experiments with MBCD–cholesterol, when no net cholesterol extraction took place. We conclude that non-lethal cholesterol depletion causes the aggregation of lipid rafts which then induces T cell signalling.     

West Nile virus entry requires cholesterol-rich membrane microdomains and is independent of alphavbeta3 integrin

West Nile virus (WNV) has been the leading cause of viral encephalitis in the United States since 1999. The endocytic processes involved in the internalization of infectious WNV by various cell types are not well characterized, and the involvement of cholesterol-rich membrane microdomains, or lipid rafts, in the life cycle of WNV has not been investigated previously. In this study, we found that the depletion of cellular cholesterol levels by brief treatment with methyl-beta-cyclodextrin resulted in a 100-fold reduction of the titers of infectious WNV released into the culture supernatant, as well as a reduction in the number of WNV genome copies in the cholesterol-depleted cells. The addition of exogenous cholesterol to cholesterol-depleted cells reversed this effect. Cholesterol depletion postinfection did not affect WNV growth, suggesting that the effect occurs at the level of WNV entry. We also showed that while WNV entry did not require alphavbeta3 integrin and focal adhesion kinase, WNV particles failed to be internalized by cholesterol-depleted cells. Finally, we showed the colocalization of the WNV envelope protein and cholera toxin B, which is internalized in a lipid raft-dependent pathway, in microdomain clusters at the plasma membrane. These data suggest that WNV utilizes lipid rafts during initial stages of internalization and that the lipid rafts may contain a factor(s) that may enhance WNV endocytosis.