RaftProt V2: understanding membrane microdomain function through lipid raft proteomes

ABSTRACTCellular membranes feature dynamic submicrometer-scale lateral membrane domainsvariously referred to as lipid rafts, membrane rafts or glycosphingolipid-enriched microdomains (GEM). In order to understand the molecular functions of lipid rafts, numerous studies have utilized various biochemical methods to isolate and examine the protein composition of membrane rafts. However, interpretation of individual raft proteomics studies are confounded by the limitations of isolation methods and the dynamic nature of rafts. Knowledge-based approaches can facilitate biological data interpretation by integrating experimental evidence from existing studies. To this end, we previously developed RaftProt (http://lipid-raft-database.di.uq.edu.au/), a searchable database of mammalian lipid raft-associated proteins. Despite being a valuable and highly used resource, improvements in search capabilities and visualisation were still needed. Here, we present RaftProt V2 (http://raftprot.org), an improved update of RaftProt, enabling interrogation and integration of datasets at the cell/tissue type and UniRef/Gene level. Besides the addition of new datasets and re-mapping of all entries to both UniProt and UniRef IDs, we have annotated the level of experimental evidence for each protein entry. The search engine now allows for multiple protein or experiment searches where correlations, interactions or overlaps can be investigated. The web-interface has been completely re-designed and offers new interactive tools for data and subset selection, correlation analysis and network visualization. Overall, RaftProt aims to advance our understanding of lipid raft function by revealing the proteomes and pathways that are associated with membrane microdomains in diverse tissue and conditions.Database URL: http://raftprot.org

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Proteomic Analysis of Lipid Rafts from RBL-2H3 Mast Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20163904 ◽

2019 ◽

Vol 20 (16) ◽

pp. 3904 ◽

Cited By ~ 2

Author(s):

Edismauro Garcia Freitas Filho ◽

Luiz Augusto Marin Jaca ◽

Lilian Cristiane Baeza ◽

Célia Maria de Almeida Soares ◽

Clayton Luiz Borges ◽

...

Keyword(s):

Mast Cells ◽

Lipid Rafts ◽

Lipid Raft ◽

Protein Composition ◽

Enrichment Analysis ◽

Functional Enrichment Analysis ◽

Cell Types ◽

Cellular Membrane ◽

Functional Enrichment ◽

Membrane Microdomains

Lipid rafts are highly ordered membrane microdomains enriched in cholesterol, glycosphingolipids, and certain proteins. They are involved in the regulation of cellular processes in diverse cell types, including mast cells (MCs). The MC lipid raft protein composition was assessed using qualitative mass spectrometric characterization of the proteome from detergent-resistant membrane fractions from RBL-2H3 MCs. Using two different post-isolation treatment methods, a total of 949 lipid raft associated proteins were identified. The majority of these MC lipid raft proteins had already been described in the RaftProtV2 database and are among highest cited/experimentally validated lipid raft proteins. Additionally, more than half of the identified proteins had lipid modifications and/or transmembrane domains. Classification of identified proteins into functional categories showed that the proteins were associated with cellular membrane compartments, and with some biological and molecular functions, such as regulation, localization, binding, catalytic activity, and response to stimulus. Furthermore, functional enrichment analysis demonstrated an intimate involvement of identified proteins with various aspects of MC biological processes, especially those related to regulated secretion, organization/stabilization of macromolecules complexes, and signal transduction. This study represents the first comprehensive proteomic profile of MC lipid rafts and provides additional information to elucidate immunoregulatory functions coordinated by raft proteins in MCs.

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Involvement of Cholesterol-Rich Lipid Rafts in Interleukin-6-Induced Neuroendocrine Differentiation of LNCaP Prostate Cancer Cells

Endocrinology ◽

10.1210/en.2003-0772 ◽

2004 ◽

Vol 145 (2) ◽

pp. 613-619 ◽

Cited By ~ 45

Author(s):

Jayoung Kim ◽

Rosalyn M. Adam ◽

Keith R. Solomon ◽

Michael R. Freeman

Keyword(s):

Prostate Cancer ◽

Signal Transduction ◽

Lipid Rafts ◽

Lipid Raft ◽

Neuron Specific Enolase ◽

Membrane Microdomains ◽

Membrane Rafts ◽

Lncap Cells ◽

Discontinuous Sucrose Gradient ◽

Triton X

Abstract IL-6 is an inflammatory cytokine that has been linked to aggressive prostate cancer (PCa). Previous studies have demonstrated that IL-6 can enhance the differentiation of PCa cells toward a neuroendocrine (NE) phenotype, a possible indicator of hormone-refractory disease. In this report, we present evidence that the mechanism of IL-6-stimulated NE differentiation employs a detergent-resistant (lipid raft) membrane compartment for signal transduction in LNCaP PCa cells. Signal transducer and activator of transcription (STAT)3, a mediator of IL-6 signaling, was rapidly phosphorylated and translocated to the nucleus in LNCaP cells treated with IL-6. Both processes were inhibited by filipin, a cholesterol-binding compound that disrupts plasma membrane lipid rafts. Isolation of Triton X-100-insoluble raft fractions from LNCaP cells by discontinuous sucrose gradient centrifugation demonstrated that the 80-kDa IL-6 receptor localized almost exclusively to the raft compartment. Although STAT3 was located predominantly in the Triton X-100-soluble subcellular fraction in exponentially growing cells, abundant phosphorylated STAT3 was detected in the raft fraction after stimulation with IL-6. Increases in expression of the NE marker, neuron-specific enolase, and neuron-specific enolase promoter activity after IL-6 treatment were reduced after membrane rafts were disrupted by filipin treatment. LNCaP cells expressed the raft-resident proteins flotillin-2 and Giα2, but notably not caveolins, the predominant structural protein present in caveolar membrane rafts in many tissues and tumor cells. These results are the first to define a role for lipid raft membrane microdomains in signal transduction mechanisms capable of promoting the NE phenotype in PCa cells, and they demonstrate that the raft compartment is capable of mediating such signals in the absence of caveolins. Our results also suggest a mechanistic role for membrane cholesterol in cell signaling events relevant to PCa progression.

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Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation

Journal of Cell Science ◽

10.1242/jcs.115.12.2603 ◽

2002 ◽

Vol 115 (12) ◽

pp. 2603-2611 ◽

Cited By ~ 7

Author(s):

Martha Triantafilou ◽

Kensuke Miyake ◽

Douglas T. Golenbock ◽

Kathy Triantafilou

Keyword(s):

Lipid Rafts ◽

Lipid Raft ◽

Cell Activation ◽

Imaging Techniques ◽

Recognition System ◽

Innate Immune ◽

Membrane Microdomains ◽

Immune Recognition ◽

Cellular Activation ◽

Signalling Molecules

The plasma membrane of cells is composed of lateral heterogeneities,patches and microdomains. These membrane microdomains or lipid rafts are enriched in glycosphingolipids and cholesterol and have been implicated in cellular processes such as membrane sorting and signal transduction. In this study we investigated the importance of lipid raft formation in the innate immune recognition of bacteria using biochemical and fluorescence imaging techniques. We found that receptor molecules that are implicated in lipopolysaccharide (LPS)-cellular activation, such as CD14, heat shock protein(hsp) 70, 90, Chemokine receptor 4 (CXCR4), growth differentiation factor 5(GDF5) and Toll-like receptor 4 (TLR4), are present in microdomains following LPS stimulation. Lipid raft integrity is essential for LPS-cellular activation, since raft-disrupting drugs, such as nystatin or MCD, inhibit LPS-induced TNF-α secretion. Our results suggest that the entire bacterial recognition system is based around the ligation of CD14 by bacterial components and the recruitment of multiple signalling molecules, such as hsp70, hsp90, CXCR4, GDF5 and TLR4, at the site of CD14-LPS ligation, within the lipid rafts.

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Palmitoylation of the Autographa californica Multicapsid Nucleopolyhedrovirus Envelope Glycoprotein GP64: Mapping, Functional Studies, and Lipid Rafts

Journal of Virology ◽

10.1128/jvi.77.11.6265-6273.2003 ◽

2003 ◽

Vol 77 (11) ◽

pp. 6265-6273 ◽

Cited By ~ 27

Author(s):

Sandy Xiaoxin Zhang ◽

Yu Han ◽

Gary W. Blissard

Keyword(s):

Cell Surface ◽

Lipid Rafts ◽

Lipid Raft ◽

Envelope Glycoprotein ◽

Cysteine Residue ◽

Autographa Californica ◽

Membrane Microdomains ◽

Sf9 Cells ◽

Wild Type ◽

Lipid Raft Microdomains

ABSTRACT Budded virions (BV) of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) contain a major envelope glycoprotein known as GP64, which was previously shown to be palmitoylated. In the present study, we used truncation and amino acid substitution mutations to map the palmitoylation site to cysteine residue 503. Palmitoylation of GP64 was not detected when Cys503 was replaced with alanine or serine. Palmitoylation-minus forms of GP64 were used to replace wild-type GP64 in AcMNPV, and these viruses were used to examine potential functions of GP64 palmitoylation in the context of the infection cycle. Analysis by immunoprecipitation and cell surface studies revealed that palmitoylation of GP64 did not affect GP64 synthesis or its transport to the cell surface in Sf9 cells. GP64 proteins lacking palmitoylation also mediated low-pH-triggered membrane fusion in a manner indistinguishable from that of wild-type GP64. Cells infected with viruses expressing palmitoylation-minus forms of GP64 produced infectious virions at levels similar to those from cells infected with wild-type AcMNPV. In combination, these data suggest that virus entry and exit in Sf9 cells were not significantly affected by GP64 palmitoylation. To determine whether GP64 palmitoylation affected the association of GP64 with membrane microdomains, the potential association of GP64 with lipid raft microdomains was examined. These experiments showed that: (i) AcMNPV-infected Sf9 cell membranes contain lipid raft microdomains, (ii) GP64 association with lipid rafts was not detected in infected Sf9 cells, and (iii) GP64 palmitoylation did not affect the apparent exclusion of GP64 from lipid raft microdomains.

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Lipid raft adhesion receptors and Syk regulate selectin-dependent rolling under flow conditions

Blood ◽

10.1182/blood-2006-04-013912 ◽

2006 ◽

Vol 108 (10) ◽

pp. 3352-3359 ◽

Cited By ~ 62

Author(s):

Claire Abbal ◽

Martine Lambelet ◽

Debora Bertaggia ◽

Carole Gerbex ◽

Manuel Martinez ◽

...

Keyword(s):

Tyrosine Kinase ◽

Lipid Rafts ◽

Lipid Raft ◽

Chelating Agents ◽

Regulatory Mechanisms ◽

Membrane Microdomains ◽

Leukocyte Rolling ◽

Flow Conditions ◽

Cell Rolling ◽

Pharmacologic Inhibitors

Abstract Selectins and their ligand P-selectin glycoprotein ligand-1 (PSGL-1) mediate leukocyte rolling along inflamed vessels. Cell rolling is modulated by selectin interactions with their ligands and by topographic requirements including L-selectin and PSGL-1 clustering on tips of leukocyte microvilli. Lipid rafts are cell membrane microdomains reported to function as signaling platforms. Here, we show that disruption of leukocyte lipid rafts with cholesterol chelating agents depleted raft-associated PSGL-1 and L-selectin and strongly reduced L-, P-, and E-selectin–dependent rolling. Cholesterol repletion reversed inhibition of cell rolling. Importantly, leukocyte rolling on P-selectin induced the recruitment of spleen tyrosine kinase (Syk), a tyrosine kinase associated to lipid raft PSGL-1. Furthermore, inhibition of Syk activity or expression, with pharmacologic inhibitors or by RNA interference, strongly reduced leukocyte rolling on P-selectin, but not on E-selectin or PSGL-1. These observations identify novel regulatory mechanisms of leukocyte rolling on selectins with a strong dependency on lipid raft integrity and Syk activity.

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Lipid raft integrity affects GABAA receptor, but not NMDA receptor modulation by psychopharmacological compounds

The International Journal of Neuropsychopharmacology ◽

10.1017/s146114571200140x ◽

2013 ◽

Vol 16 (6) ◽

pp. 1361-1371 ◽

Cited By ~ 16

Author(s):

Caroline Nothdurfter ◽

Sascha Tanasic ◽

Barbara Di Benedetto ◽

Manfred Uhr ◽

Eva-Maria Wagner ◽

...

Keyword(s):

Nmda Receptor ◽

Lipid Rafts ◽

Gabaa Receptor ◽

Lipid Raft ◽

Tricyclic Antidepressant ◽

Membrane Microdomains ◽

Cholesterol Depletion ◽

Receptor Modulation ◽

Gabaa Receptor Subunits ◽

Triton X

Abstract Lipid rafts have been shown to play an important role for G-protein mediated signal transduction and the function of ligand-gated ion channels including their modulation by psychopharmacological compounds. In this study, we investigated the functional significance of the membrane distribution of NMDA and GABAA receptor subunits in relation to the accumulation of the tricyclic antidepressant desipramine (DMI) and the benzodiazepine diazepam (Diaz). In the presence of Triton X-100, which allowed proper separation of the lipid raft marker proteins caveolin-1 and flotillin-1 from the transferrin receptor, all receptor subunits were shifted to the non-raft fractions. In contrast, under detergent-free conditions, NMDA and GABAA receptor subunits were detected both in raft and non-raft fractions. Diaz was enriched in non-raft fractions without Triton X-100 in contrast to DMI, which preferentially accumulated in lipid rafts. Impairment of lipid raft integrity by methyl-β-cyclodextrine (MβCD)-induced cholesterol depletion did not change the inhibitory effect of DMI at the NMDA receptor, whereas it enhanced the potentiating effect of Diaz at the GABAA receptor at non-saturating concentrations of GABA. These results support the hypothesis that the interaction of benzodiazepines with the GABAA receptor likely occurs outside of lipid rafts while the antidepressant DMI acts on ionotropic receptors both within and outside these membrane microdomains.

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Vav1/Rac-dependent actin cytoskeleton reorganization is required for lipid raft clustering in T cells

The Journal of Cell Biology ◽

10.1083/jcb.200107080 ◽

2001 ◽

Vol 155 (3) ◽

pp. 331-338 ◽

Cited By ~ 168

Author(s):

Martin Villalba ◽

Kun Bi ◽

Fernando Rodriguez ◽

Yoshihiko Tanaka ◽

Stephen Schoenberger ◽

...

Keyword(s):

T Cells ◽

Actin Cytoskeleton ◽

Lipid Rafts ◽

Lipid Raft ◽

Large Scale ◽

Actin Polymerization ◽

Dominant Negative ◽

Membrane Microdomains ◽

Jurkat T Cells ◽

Cytoskeleton Reorganization

Formation of the immunological synapse (IS) in T cells involves large scale molecular movements that are mediated, at least in part, by reorganization of the actin cytoskeleton. Various signaling proteins accumulate at the IS and are localized in specialized membrane microdomains, known as lipid rafts. We have shown previously that lipid rafts cluster and localize at the IS in antigen-stimulated T cells. Here, we provide evidence that lipid raft polarization to the IS depends on an intracellular pathway that involves Vav1, Rac, and actin cytoskeleton reorganization. Thus, lipid rafts did not translocate to the IS in Vav1-deficient (Vav1−/−) T cells upon antigen stimulation. Similarly, T cell receptor transgenic Jurkat T cells also failed to translocate lipid rafts to the IS when transfected with dominant negative Vav1 mutants. Raft polarization induced by membrane-bound cholera toxin cross-linking was also abolished in Jurkat T cells expressing dominant negative Vav1 or Rac mutants and in cells treated with inhibitors of actin polymerization. However, Vav overexpression that induced F-actin polymerization failed to induce lipid rafts clustering. Therefore, Vav is necessary, but not sufficient, to regulate lipid rafts clustering and polarization at the IS, suggesting that additional signals are required.

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Bcr-Abl Inhibition by Imatinib Restores Lyn Trafficking to Lipid Rafts and Promotes CXCR4 Surface Expression of CML Cells in BM Microenvironment

Blood ◽

10.1182/blood.v112.11.3188.3188 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3188-3188

Author(s):

Yoko Tabe ◽

Linhua Jin ◽

Naoki Ichikawa ◽

Marina Konopleva ◽

Michael Andreeff ◽

...

Keyword(s):

Cell Surface ◽

Tyrosine Kinase ◽

Lipid Rafts ◽

Lipid Raft ◽

Surface Expression ◽

Culture Conditions ◽

Cxcr4 Expression ◽

Membrane Microdomains ◽

Protein Levels ◽

Significant Difference

Abstract Chronic myeloid leukemia (CML) is driven by the constitutively activated Bcr-Abl tyrosine kinase, which causes deficiency in CXCR4-mediated migration of CML cells to bone marrow (BM) stroma. We have recently demonstrated that exposure of CML cells to imatinib under stromal co-cultures results in increased CXCR4 surface expression, enhanced migration of CML cells towards stromal cell layers and non-pharmacological resistance to imatinib (Jin, Mol Cancer Ther2008;7:48). Lipid rafts are plasma membrane microdomains, highly enriched in cholesterol, sphingolipids and in signaling molecules, which act as signal transduction platforms for a variety of intracellular processes. Lyn is a Src-family tyrosine kinase that is a downstream target of Bcr-Abl, and frequently localizes in lipid raft fractions. Binding to Bcr-Abl results in the constitutive activation of Lyn which impairs SDF-1 Ptasznik, J Exp Med2002;196:667). In this study, we investigated the effects of the tyrosine kinase inhibitor imatinib on the localization of Lyn in the lipid raft structures of CML cells under conditions mimicking the BM microenvironment. Imatinib treatment significantly increased cell surface CXCR4 expression levels in KBM5 CML cells only under mesenchymal stem cell (MSC) co-culture conditions as determined by FACS analysis (p<0.01). However, no significant difference in total CXCR4 protein levels was observed in control and imatinib/MSC co-cultured KBM5 cells by immunoblotting. These findings were confirmed by confocal microscopic analyses, whereby direct coculture of imatinib-treated KBM5 cells with MSC resulted in the increased expression of CXCR4 protein levels on the KBM5 cell surface without change in intracellular protein levels. In turn, KBM5 cells treated with imatinb in the absence of MSC, or co-cultured with MSC alone, showed no significant upregulation of surface CXCR4 expression. Analysis of lipid raft fractions using discontinuous sucrose density gradient fractionation demonstrated that Lyn strongly localized to lipid rafts in imatinib(+)/MSC(+) KBM5 cells compared to control KBM5 cells (5.2-fold increase in the ratio of Lyn to the raft marker flotillin-1). On the contrary, imatinib(+)/MSC(−) or imatinib(−)/MSC(+) conditioned KBM5 cells expressed similar levels of Lyn/flotillin in raft fractions. No significant difference in the levels of total or phosphorylated (Tyr396 and Tyr507) Lyn in whole cell lysates was detected by immunoblotting under all tested conditions.In conclusion, these findings demonstrate, for the first time, that Bcr-Abl oncoprotein inhibits Lyn trafficking to lipid raft microdomains in CML cells. Inhibition of Bcr-Abl by imatinib under stromal co-culture conditions promotes Lyn localization to the lipid rafts which in turn results in increased CXCR4 cell surface expression. These findings indicate that blockade of Lyn expression may ameliorate microenvironment-mediated resistance to tyrosine kinase inhibitors in CML.

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Lipid Rafts: Linking Alzheimer's Amyloid-βProduction, Aggregation, and Toxicity at Neuronal Membranes

International Journal of Alzheimer s Disease ◽

10.4061/2011/603052 ◽

2011 ◽

Vol 2011 ◽

pp. 1-14 ◽

Cited By ~ 34

Author(s):

Jo V. Rushworth ◽

Nigel M. Hooper

Keyword(s):

Glutamate Receptors ◽

Lipid Rafts ◽

Lipid Raft ◽

Cell Signalling ◽

Cellular Prion Protein ◽

Membrane Microdomains ◽

Synaptic Dysfunction ◽

Memory Impairments ◽

Neuronal Membranes ◽

Neuronal Functions

Lipid rafts are membrane microdomains, enriched in cholesterol and sphingolipids, into which specific subsets of proteins and lipids partition, creating cell-signalling platforms that are vital for neuronal functions. Lipid rafts play at least three crucial roles in Alzheimer's Disease (AD), namely, in promoting the generation of the amyloid-β(Aβ) peptide, facilitating its aggregation upon neuronal membranes to form toxic oligomers and hosting specific neuronal receptors through which the AD-related neurotoxicity and memory impairments of the Aβoligomers are transduced. Recent evidence suggests that Aβoligomers may exert their deleterious effects through binding to, and causing the aberrant clustering of, lipid raft proteins including the cellular prion protein and glutamate receptors. The formation of these pathogenic lipid raft-based platforms may be critical for the toxic signalling mechanisms that underlie synaptic dysfunction and neuropathology in AD.

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Membrane lipid rafts are required for D2 dopamine receptor signaling

European Psychiatry ◽

10.1016/s0924-9338(11)72615-3 ◽

2011 ◽

Vol 26 (S2) ◽

pp. 910-910 ◽

Cited By ~ 1

Author(s):

D. Sibley ◽

L. Hazelwood ◽

R. Roof ◽

R.B. Free ◽

Y. Han ◽

...

Keyword(s):

Dopamine Receptor ◽

Lipid Rafts ◽

Lipid Raft ◽

Gradient Centrifugation ◽

Membrane Lipid ◽

Receptor Protein ◽

Membrane Microdomains ◽

Membrane Cholesterol ◽

Camp Accumulation ◽

D2 Dopamine Receptor

IntroductionLipid rafts are specialized membrane microdomains enriched in cholesterol and sphingolipids and are important in the organization of receptor-protein complexes and the regulation of signaling.Objective/aimsGiven the emerging significance of lipids with respect to receptor structure and activation, we investigated the role of lipid rafts and membrane cholesterol on D2 dopamine receptor (DAR) signaling. As the D2 DAR is the molecular target for all antipsychotic drugs, more information about its signaling may help refine therapeutics for schizophrenia.MethodsD2 DAR constructs were expressed in HEK293T cells. Sucrose density fractionation resolved lipid rafts from other membrane components. Methyl-β-cyclodextrin (MCD) was used to deplete membrane cholesterol and to disrupt lipid rafts.ResultsDetergent solubilization followed by sucrose gradient centrifugation resolved lipid rafts from heavier membrane fractions. The D2 DAR was equally distributed amongst both the lipid raft and heavier membrane fractions. Pretreatment with MCD, however, eliminated both lipid raft markers and the D2 DAR from lipid raft fractions, although the receptor was still found in heavier membrane fractions. We also found that MCD treatment abolished D2 DAR-mediated inhibition of cAMP accumulation. In contrast D1 DAR-stimulated cAMP accumulation was unaffected by MCD treatment.ConclusionsOur current results show that the D2 DAR is distributed in multiple membrane microdomains, including cholesterol-rich lipid rafts. We found that extraction of cholesterol disrupted lipid rafts and also an eliminated D2 DAR-mediated signaling. Thus, we hypothesize that lipid rafts are critical for D2 DAR signaling to occur.

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