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

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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Monosialoganglioside GM1 Deficiency Inhibits the Neurotrophic Effects of GDNF by Disrupting Lipid Raft Assembly

10.21203/rs.3.rs-1100361/v1 ◽

2021 ◽

Author(s):

Shimin Jiang ◽

Tai Zhou ◽

Kejia Zhang ◽

Yao Zhou ◽

Zhongcheng Wang ◽

...

Keyword(s):

Lipid Rafts ◽

Lipid Raft ◽

Gradient Centrifugation ◽

Membrane Microdomains ◽

Western Blot Assay ◽

Caveolin 1 ◽

Downstream Signaling ◽

Proliferation And Differentiation ◽

Lipid Raft Microdomains ◽

Triton X

Abstract Recent studies have shown that monosialoganglioside GM1 deficiency can inhibit the signal transduction process of glial cell line-derived neurotrophic factor (GDNF), which plays an important role in the pathogenesis of Parkinson's disease (PD). However, its specific mechanism still needs to be explored. We inhibited the expression of GM1 by treating cells with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). CCK-8 assay, EdU cell proliferation assay and Western blot assay were used to evaluate the effect of GM1 deficiency on the proliferation and differentiation of SH-SY5Y cells induced by GDNF and on the GDNF-RET signaling pathway. Lipid rafts were isolated by Triton X-100 solubilization and OptiPrepTM density gradient centrifugation. The alterations of lipid raft assembly and the translocation of RET into lipid rafts were evaluated after PDMP treatment. We found that PDMP treatment inhibited the proliferation and differentiation of SH-SY5Y cells induced by GDNF and reduced the phosphorylation of RET and its downstream signaling molecules Erk and Akt. In addition, after PDMP treatment, caveolin-1 and flotillin-1, the prototypical markers of lipid rafts, diffused from lipid rafts to non-lipid raft microdomains, and GDNF-induced RET translocation into lipid rafts was also reduced. These alterations could be partially reversed by adding exogenous GM1. Our results suggest that ganglioside GM1 deficiency could compromise the neurotrophic effects and signals downstream of GDNF by altering the assembly of lipid raft membrane microdomains.

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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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Spatiotemporal Analysis of Differential Akt Regulation in Plasma Membrane Microdomains

Molecular Biology of the Cell ◽

10.1091/mbc.e08-05-0449 ◽

2008 ◽

Vol 19 (10) ◽

pp. 4366-4373 ◽

Cited By ~ 97

Author(s):

Xinxin Gao ◽

Jin Zhang

Keyword(s):

Plasma Membrane ◽

Growth Factor ◽

Lipid Rafts ◽

Fluorescent Proteins ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Spatiotemporal Analysis ◽

Membrane Microdomains ◽

Cholesterol Depletion ◽

Growth Factor Signaling

As a central kinase in the phosphatidylinositol 3-kinase pathway, Akt has been the subject of extensive research; yet, spatiotemporal regulation of Akt in different membrane microdomains remains largely unknown. To examine dynamic Akt activity in membrane microdomains in living cells, we developed a specific and sensitive fluorescence resonance energy transfer-based Akt activity reporter, AktAR, through systematic testing of different substrates and fluorescent proteins. Targeted AktAR reported higher Akt activity with faster activation kinetics within lipid rafts compared with nonraft regions of plasma membrane. Disruption of rafts attenuated platelet-derived growth factor (PDGF)-stimulated Akt activity in rafts without affecting that in nonraft regions. However, in insulin-like growth factor-1 (IGF)-1 stimulation, Akt signaling in nonraft regions is dependent on that in raft regions. As a result, cholesterol depletion diminishes Akt activity in both regions. Thus, Akt activities are differentially regulated in different membrane microdomains, and the overall activity of this oncogenic pathway is dependent on raft function. Given the increased abundance of lipid rafts in some cancer cells, the distinct Akt-activating characteristics of PDGF and IGF-1, in terms of both effectiveness and raft dependence, demonstrate the capabilities of different growth factor signaling pathways to transduce differential oncogenic signals across plasma membrane.

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Dynamics of putative raft-associated proteins at the cell surface

The Journal of Cell Biology ◽

10.1083/jcb.200312170 ◽

2004 ◽

Vol 165 (5) ◽

pp. 735-746 ◽

Cited By ~ 329

Author(s):

Anne K. Kenworthy ◽

Benjamin J. Nichols ◽

Catha L. Remmert ◽

Glenn M. Hendrix ◽

Mukesh Kumar ◽

...

Keyword(s):

Cell Surface ◽

Lipid Rafts ◽

Long Range ◽

Dominant Factor ◽

Membrane Microdomains ◽

Cholesterol Depletion ◽

Biochemical Fractionation ◽

Associated Proteins ◽

Raft Domains

Lipid rafts are conceptualized as membrane microdomains enriched in cholesterol and glycosphingolipid that serve as platforms for protein segregation and signaling. The properties of these domains in vivo are unclear. Here, we use fluorescence recovery after photobleaching to test if raft association affects a protein's ability to laterally diffuse large distances across the cell surface. The diffusion coefficients (D) of several types of putative raft and nonraft proteins were systematically measured under steady-state conditions and in response to raft perturbations. Raft proteins diffused freely over large distances (>4 μm), exhibiting Ds that varied 10-fold. This finding indicates that raft proteins do not undergo long-range diffusion as part of discrete, stable raft domains. Perturbations reported to affect lipid rafts in model membrane systems or by biochemical fractionation (cholesterol depletion, decreased temperature, and cholesterol loading) had similar effects on the diffusional mobility of raft and nonraft proteins. Thus, raft association is not the dominant factor in determining long-range protein mobility at the cell surface.

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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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The voltage-gated sodium channel β2 subunit associates with lipid rafts by S-palmitoylation

Journal of Cell Science ◽

10.1242/jcs.252189 ◽

2021 ◽

Vol 134 (6) ◽

Author(s):

Eric Cortada ◽

Robert Serradesanferm ◽

Ramon Brugada ◽

Marcel Verges

Keyword(s):

Sodium Channel ◽

Lipid Rafts ◽

Lipid Raft ◽

Cholesterol Depletion ◽

Slight Reduction ◽

Apical Surface ◽

Voltage Gated Sodium Channel ◽

Α Subunit ◽

Voltage Gated ◽

Partial Overlap

ABSTRACT The voltage-gated sodium channel is critical for cardiomyocyte function. It consists of a protein complex comprising a pore-forming α subunit and associated β subunits. In polarized Madin–Darby canine kidney cells, we show evidence by acyl-biotin exchange that β2 is S-acylated at Cys-182. Interestingly, we found that palmitoylation increases β2 association with detergent-resistant membranes. β2 localizes exclusively to the apical surface. However, depletion of plasma membrane cholesterol, or blocking intracellular cholesterol transport, caused mislocalization of β2, as well as of the non-palmitoylable C182S mutant, to the basolateral domain. Apical β2 did not undergo endocytosis and displayed limited diffusion within the plane of the membrane; such behavior suggests that, at least in part, it is cytoskeleton anchored. Upon acute cholesterol depletion, its mobility was greatly reduced, and a slight reduction was also measured as a result of lack of palmitoylation, supporting β2 association with cholesterol-rich lipid rafts. Indeed, lipid raft labeling confirmed a partial overlap with apical β2. Although β2 palmitoylation was not required to promote surface localization of the α subunit, our data suggest that it is likely implicated in lipid raft association and the polarized localization of β2.

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RaftProt V2: understanding membrane microdomain function through lipid raft proteomes

10.1101/286039 ◽

2018 ◽

Author(s):

Ahmed Mohamed ◽

Anup Shah ◽

David Chen ◽

Michelle M. Hill

Keyword(s):

Experimental Evidence ◽

Lipid Rafts ◽

Lipid Raft ◽

Protein Composition ◽

Biological Data ◽

Tissue Type ◽

Membrane Microdomains ◽

Membrane Rafts ◽

Link Type ◽

Membrane Microdomain

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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