The Facultative Role of Lipid Rafts and Membrane Microdomains in Controlling the Assembly of the Store-Operated Channel Complex

Abstract Sulfatide (galactocylceramide 3′-sulfate) is a sulfated glycosphingolipid expressed on the surfaces of erythrocytes, leukocytes, platelets and a variety other cells, that is known to interact with several cell adhesion molecules involved in hemostasis, including von Willebrand factor (VWF), laminin, thrombospondin, P-selectin and β2-glycoprotein I. Because these ligands are involved in many platelet adhesive interactions, we hypothesize that membrane sulfatide plays an important role in these processes. To examine this, we have cloned and purified a sulfatide-specific single-chain variable fragment (scFv) antibody from a phage-display library constructed from mRNA taken from the lymphocytes of patients with systemic lupus erythematosis. This scFv, PA38, specifically bound sulfatide, and did not react with the related sphingolipids cerebroside, ceramide, or sphingomyelin, or the phospholipids phosphatidylserine, phosphatidylcholine, or phosphatidylethanolamine. Using this tool, we examined the role of sulfatide in platelet function. We observed that PA38 dose-dependently (at 5 and 10 μg/ml) inhibited the aggregation of human platelets induced by either collagen or ADP. A control scFv produced in a similar manner had no effect. Furthermore, PA38 delayed platelet plug formation by 23 sec (with collagen-ADP agonist) and 46 sec (with collagen-epinephrine) in whole blood from normal human donors, as measured in a platelet function analyzer, PFA-100 (Dade Behring). Further, to verify that this was a sulfatide-specific effect, we compared collagen-induced platelet aggregation in normal mice to that of mice deficient in cerebroside sulfotransferase (CST)—a critical enzyme in the sulfatide synthetic pathway. The CST−/− mice fail to express sulfatide on the cell surface, and displayed defective platelet aggregation. Consistent with this, the PA38 also significantly inhibited collagen-induce platelet aggregation in wild-type mice. Given the importance of lipid rafts in signaling and adhesive processes, we looked for the localization of sulfatide in these membrane microdomains. Indeed, we found that sulfatide is enriched in lipid rafts suggesting a role for sulfatide in lipid-raft mediated events. Thus, we provide evidence for a key role of a membrane lipid, sulfatide in the adhesive interactions involved in platelet function. With one notable exception, the key adhesive roles in platelet-platelet interaction have all previously been assigned to proteins.

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Modulation of Tissue Factor-Factor VIIa Signaling by Lipid Rafts and Caveolae.

Blood ◽

10.1182/blood.v108.11.1744.1744 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1744-1744

Author(s):

Vineet Awasthi ◽

Samir Mandal ◽

Veena Papanna ◽

L. Vijaya Mohan Rao ◽

Usha Pendurthi

Keyword(s):

Cell Signaling ◽

Lipid Rafts ◽

G Proteins ◽

Intracellular Signaling ◽

Factor Viia ◽

Membrane Microdomains ◽

Caveolin 1 ◽

Intracellular Signaling Pathways ◽

Plasma Membrane Microdomains

Abstract Tissue factor (TF) is a cellular receptor for clotting factor VIIa (VIIa) and the formation of TF-VIIa complexes on cell surfaces not only triggers the coagulation cascade but also transduces cell signaling via activation of protease-activated receptors (PARs), particularly PAR2. Although a number of recent studies provide valuable information on intracellular signaling pathways that are activated by TF-VIIa, the role of various cell surface components in mediating the interaction of TF-VIIa with PARs, and the subsequent signal transmittance are unknown. Unlike thrombin and trypsin, VIIa has to bind to its cellular receptor (TF) to activate PARs. The inability of TF-VIIa to effectively activate Ca2+ signaling and failure to desensitize the signaling to subsequently added trypsin suggest that the TF-VIIa is a poor activator of PAR2. Despite this, a number of studies have shown that VIIa is as effective as trypsin or PAR2 agonist peptide in activating intracellular signaling pathways and gene expression in cells expressing TF. Although the potential mechanism for this phenomenon is unknown, compartmentalization of TF, PAR2, and G-proteins in plasma membrane microdomains could facilitate a robust TF-VIIa-induced PAR2-mediated cell signaling. Although certain G-protein coupled receptors and G-proteins are known to be segregated into specialized membrane microdomains, lipid rafts and caveolae, little is known whether PARs are segregated into lipid rafts and caveolae, and how such segregation might influence their activation by TF-VIIa and the subsequent coupling to G-proteins. To obtain answers to some of these questions, in the present study, we have characterized TF and PAR2 distribution on tumor cell surfaces and investigated the role of lipid raft/caveolae in modulating the TF-VIIa signaling in tumor cells. Detergent extraction of cells followed by fractionation on sucrose gradient centrifugation showed that TF and PAR2 were distributed both in lipid rafts (low-density) and soluble fractions. Immunofluorescence confocal microscopy revealed that TF at the cell surface is localized in discrete plasma membrane microdomains, and colocalized with caveolin-1, a structural integral protein of caveolae, indicating caveolar localization of TF. Similar to TF, PAR2 also displayed significant punctuate staining and colocalization with caveloin-1. Further, a substantial fraction of TF and PAR2 was colocalized in caveolae. Disruption of lipid rafts/caveolae by ß-methyl cyclodextrin or filipin treatments reduced TF association with PAR2 in lipid rafts and caveolar fractions and impaired the TF-VIIa-induced cell signaling (PI hydrolysis and IL-8 gene expression). Additional studies showed that both mßCD and filipin treatments specifically impaired TF-VIIa cleavage of PAR2 and but had no significant effect on trypsin cleavage of PAR2. Disruption of caveolae with caveolin-1 silencing had no effect on the TF-VIIa coagulant activity but inhibited the TF-VIIa-induced cell signaling. In summary, the data presented herein demonstrate that TF localization at the cell membrane could influence different functions of TF differently. While caveolar localization of TF had no influence in propagating the procoagulant activity of TF, it is essential in supporting the TF-VIIa-induced cell signaling.

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Role for lipid rafts in regulating interleukin-2 receptor signaling

Blood ◽

10.1182/blood.v98.5.1489 ◽

2001 ◽

Vol 98 (5) ◽

pp. 1489-1497 ◽

Cited By ~ 92

Author(s):

Mina D. Marmor ◽

Michael Julius

Keyword(s):

Lipid Rafts ◽

Interleukin 2 ◽

Phosphatidyl Inositol ◽

Membrane Microdomains ◽

Lipid Microdomains ◽

Interleukin 2 Receptor ◽

Lipid Environment ◽

Plasma Membrane Microdomains ◽

Nonionic Detergents

Lipid rafts are plasma membrane microdomains characterized by a unique lipid environment enriched in gangliosides and cholesterol, leading to their insolubility in nonionic detergents. Many receptors are constitutively or inducibly localized in lipid rafts, which have been shown to function as platforms coordinating the induction of signaling pathways. In this report, the first evidence is provided for a role of these lipid microdomains in regulating interleukin-2 receptor (IL-2R) signaling. It is demonstrated that antibody- or ligand-mediated immobilization of components of lipid rafts, glycosyl-phosphatidyl-inositol–anchored proteins, and the GM1 ganglioside, respectively, inhibit IL-2–induced proliferation in T cells. IL-2Rα is shown to be constitutively enriched in rafts and further enriched in the presence of immobilized anti–Thy-1. In contrast, IL-2Rβ and IL-2Rγ, as well as JAK1 and JAK3, are found in soluble membrane fractions, and their localization is not altered by anti–Thy-1. IL-2–mediated heterotrimerization of IL-2R chains is shown to occur within soluble membrane fractions, exclusively, as is the activation of JAK1 and JAK3. As predicted by these results, the disruption of lipid raft integrity did not impair IL-2–induced signaling. Thus, the sequestration of IL-2Rα within lipid microdomains restricts its intermolecular interactions and regulates IL-2R signaling through impeding its association with IL-2Rβ and IL-2Rγ.

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A critical role of lipid rafts in the organization of a key FcγRIIa-mediated signaling pathway in human platelets

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613449 ◽

2003 ◽

Vol 89 (02) ◽

pp. 318-330 ◽

Cited By ~ 35

Author(s):

Stéphane Bodin ◽

Cécile Viala ◽

Ashraf Ragab ◽

Bernard Payrastre

Keyword(s):

Lipid Rafts ◽

Critical Role ◽

Adenosine Diphosphate ◽

Human Platelets ◽

Temporal Organization ◽

Membrane Microdomains ◽

Cross Linking ◽

Dependent Manner ◽

Signaling Complex

SummaryThe involvement of platelet FcγRIIa in heparin-associated thrombocytopenia (HIT) is now well established. However, the precise sequence of molecular events initiated by FcγRIIa cross-linking in platelets remains partly characterized. We investigated here the role of lipid rafts in the spatio-temporal organization of the FcγRIIa-dependent signaling events. Upon cross-linking, FcγRIIa relocated in rafts where the kinase Lyn and the adapter LAT were among the major phosphotyrosyl proteins. Upon stimulation by HIT sera, the second messenger phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) accumulated in rafts in a P2Y12 adenosine diphosphate (ADP) recep- tor-dependent manner. PtdIns(3,4,5)P3 was then essential to specifically recruit phospholipase Cγ2 (PLCγ2) to these membrane microdomains. Controlled disruption of rafts by methyl γ-cyclodextrin reversibly abolished PtdIns(3,4,5)P3 production, PLC activation and platelet responses induced by FcγRIIa cross-linking without affecting the tyrosine phosphorylation events. This work demonstrates that platelet rafts are essential for the integration of a key signaling complex leading to the rapid production of PtdIns(3,4,5)P3 and in turn PLCγ2 activation during HIT.

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Caveolins, caveolae, and lipid rafts in cellular transport, signaling, and disease

Biochemistry and Cell Biology ◽

10.1139/o03-071 ◽

2004 ◽

Vol 82 (1) ◽

pp. 129-144 ◽

Cited By ~ 149

Author(s):

Andrew F.G Quest ◽

Lisette Leyton ◽

Mario Párraga

Keyword(s):

Lipid Rafts ◽

Structural Integrity ◽

Membrane Microdomains ◽

Cellular Transport ◽

Knock Out ◽

Caveolin 1 ◽

Membrane Compartments ◽

Knock Out Mice ◽

And Function

Caveolae were initially described some 50 years ago. For many decades, they remained predominantly of interest to structural biologists. The identification of a molecular marker for these domains, caveolin, combined with the possibility to isolate such cholesterol- and sphingolipid-rich regions as detergent-insoluble membrane complexes paved the way to more rigorous characterization of composition, regulation, and function. Experiments with knock-out mice for the caveolin genes clearly demonstrate the importance of caveolin-1 and -3 in formation of caveolae. Nonetheless, detergent-insoluble domains are also found in cells lacking caveolin expression and are referred to here as lipid rafts. Caveolae and lipid rafts were shown to represent membrane compartments enriched in a large number of signaling molecules whose structural integrity is essential for many signaling processes. Caveolin-1 is an essential structural component of cell surface caveolae, important for regulating trafficking and mobility of these vesicles. In addition, caveolin-1 is found at many other intracellular locations. Variations in subcellular localization are paralleled by a plethora of ascribed functions for this protein. Here, more recent data addressing the role of caveolin-1 in cellular signaling and the development of diseases like cancer will be preferentially discussed.Key words: caveolae, rafts, membrane microdomains, caveolins, signal transduction, disease, cancer.

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G-protein coupled receptors in lipid rafts and caveolae: how, when and why do they go there?

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0320325 ◽

2004 ◽

Vol 32 (2) ◽

pp. 325-338 ◽

Cited By ~ 257

Author(s):

B Chini ◽

M Parenti

Keyword(s):

G Protein ◽

Lipid Rafts ◽

G Protein Coupled Receptors ◽

Fine Tuning ◽

Membrane Microdomains ◽

Signalling Molecules ◽

Cell Functions ◽

Experimental Approaches ◽

G Protein Coupled

This review describes the advances in our understanding of the role of G-protein coupled receptor (GPCR) localisation in membrane microdomains known as lipid rafts and caveolae. The growing interest in these specialised regions is due to the recognition that they are involved in the regulation of a number of cell functions, including the fine-tuning of various signalling molecules. As a number of GPCRs have been found to be enriched in lipid rafts and/or caveolae by means of different experimental approaches, we first discuss the pitfalls and uncertainties related to the use of these different procedures. We then analyse the addressing signals that drive and/or stabilise GPCRs in lipid rafts and caveolae, and explore the role of rafts/caveolae in regulating GPCR trafficking, particularly in receptor exo- and endocytosis. Finally, we review the growing evidence that lipid rafts and caveolae participate in the regulation of GPCR signalling by affecting both signalling selectivity and coupling efficacy.

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Role of Stromal Microenvironment In Non-Pharmacological Resistance of CML to Tyrosine Kinase Inhibitors through Lyn/CXCR4 Interactions In Lipid Rafts.

Blood ◽

10.1182/blood.v116.21.3390.3390 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3390-3390

Author(s):

Yoko Tabe ◽

Linhua Jin ◽

Zhou Yixin ◽

Naoki Ichikawa ◽

Kazuhisa Iwabuchi ◽

...

Keyword(s):

Cell Surface ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Lipid Rafts ◽

Lipid Raft ◽

Stromal Cells ◽

Kinase Inhibitors ◽

Surface Expression ◽

Membrane Microdomains

Abstract Abstract 3390 In chronic myeloid leukemia (CML), the mechanisms of resistance to tyrosine kinase inhibitors (TKIs) beyond the Bcr-Abl mutations are not well understood. We have previously reported that TKI imatinib induces cell-surface expression of the chemokine receptor CXCR4, which results in enhanced migration towards CXCL12-producing BM stromal cells, promotes cell quiescence and development of the microenvironment-mediated, non-pharmacological drug resistance (Jin, Mol Cancer Ther 2008;7:48). Bcr-Abl tyrosine kinase directly activates Src-related kinase Lyn known to frequently localize in lipid raft plasma membrane microdomains and interact with CXCL12/CXCR4 signaling and is directly activated by p210Bcr-Abl. In this study, we investigated the effects of TKIs on the localization and interaction of CXCR4 and Lyn in the lipid rafts, and the role of lipid rafts as the signal transduction platform for CML cell migration. Confocal microscopy and discontinuous sucrose density gradient fractionation demonstrated that in CML cells CXCR4 primarily localized in the non-raft cell surface regions, while Lyn was present both in the lipid raft and non-raft fractions. In turn, the active, phosphorylated form (p-)LynTyr396 is present within the lipid rafts, while inactive p-LynTyr507 in non-raft fractions. Imatinib treatment under co-culture with mesenchymal stem cells (MSC) induced CXCR4 clustering in lipid raft fractions, which was directly co-immunoprecipitaed with Lyn. Under these culture conditions, imatinib repressed p-LynTyr507, but failed to deplete p-LynTyr396. Knock-down of Lyn by siRNA, Src inhibitor treatment, or lipid raft destruction by methyl-b cyclodextrin (MbCD) abrogated imatinib-induced KBM5 migration to MSCs and CXCL12 without affecting CXCR4 surface expression. Consistent with its effects on Src, dual Src/Abl kinase inhibitor dasatinib induced significantly less migration of CML cells to CXCL12 compared with imatinib or nilotinib (p =0.04). In summary, our data indicate that stromal cells interfere with inhibitory effects of TKI on active Lyn (p-Lyn)Tyr396 in CML cells and promote clustering of CXCR4 in lipid rafts where it co-localizes with p-LynTyr396 and facilitates migration of CML cells to the MSC monolayer. Lipid raft disruption by cholesterol depletion inhibit CML cells migration, suggesting that lipid rafts represent one of the key signaling modules responsible for interactions of CML cells with cells of BM niche. We propose that pharmacological disruption of lipid rafts may eliminate BM-resident CML cells through interference with microenvironment-mediated resistance. Disclosures: No relevant conflicts of interest to declare.

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Role of Lipid Rafts in Hematopoietic Stem Cells Homing, Mobilization, Hibernation, and Differentiation

Cells ◽

10.3390/cells8060630 ◽

2019 ◽

Vol 8 (6) ◽

pp. 630 ◽

Cited By ~ 3

Author(s):

Munther Alomari ◽

Dana Almohazey ◽

Sarah Ameen Almofty ◽

Firdos Alam Khan ◽

Mohammad Al hamad ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Lipid Rafts ◽

Transforming Growth Factor ◽

Critical Role ◽

Transforming Growth Factor Β ◽

Lymphoid Cells ◽

Membrane Microdomains ◽

Hematopoietic Stem

Hematopoietic stem cells (HSCs) are multipotent, self-renewing cells that can differentiate into myeloid or lymphoid cells. The mobilization and differentiation processes are affected by the external environment, such as extracellular matrix and soluble molecules in the niche, where the lipid rafts (LRs) of the HSCs act as the receptors and control platforms for these effectors. LRs are membrane microdomains that are enriched in cholesterol, sphingolipid, and proteins. They are involved in diverse cellular processes including morphogenesis, cytokinesis, signaling, endocytic events, and response to the environment. They are also involved in different types of diseases, such as cancer, Alzheimer’s, and prion disease. LR clustering and disruption contribute directly to the differentiation, homing, hibernation, or mobilization of HSCs. Thus, characterization of LR integrity may provide a promising approach to controlling the fate of stem cells for clinical applications. In this review, we show the critical role of LR modification (clustering, disruption, protein incorporation, and signal responding) in deciding the fate of HSCs, under the effect of soluble cytokines such as stem cell factor (SCF), transforming growth factor- β (TGF-β), hematopoietic-specific phospholipase Cβ2 (PLC-β2), and granulocyte colony-stimulating factor (G-CSF).

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Overproduction of Flotillin Influences Cell Differentiation and Shape in Bacillus subtilis

mBio ◽

10.1128/mbio.00719-13 ◽

2013 ◽

Vol 4 (6) ◽

Cited By ~ 28

Author(s):

Benjamin Mielich-Süss ◽

Johannes Schneider ◽

Daniel Lopez

Keyword(s):

Signal Transduction ◽

Bacillus Subtilis ◽

Cell Differentiation ◽

Lipid Rafts ◽

Eukaryotic Cells ◽

Membrane Microdomains ◽

Functional Link ◽

Working Model ◽

Physiological Alterations

ABSTRACTBacteria organize many membrane-related signaling processes in functional microdomains that are structurally and functionally similar to the lipid rafts of eukaryotic cells. An important structural component of these microdomains is the protein flotillin, which seems to act as a chaperone in recruiting other proteins to lipid rafts to facilitate their interaction. In eukaryotic cells, the occurrence of severe diseases is often observed in combination with an overproduction of flotillin, but a functional link between these two phenomena is yet to be demonstrated. In this work, we used the bacterial modelBacillus subtilisas a tractable system to study the physiological alterations that occur in cells that overproduce flotillin. We discovered that an excess of flotillin altered specific signal transduction pathways that are associated with the membrane microdomains of bacteria. As a consequence of this, we detected significant defects in cell division and cell differentiation. These physiological alterations were in part caused by an unusual stabilization of the raft-associated protease FtsH. This report opens the possibility of using bacteria as a working model to better understand fundamental questions related to the functionality of lipid rafts.IMPORTANCEThe identification of signaling platforms in the membrane of bacteria that are functionally and structurally equivalent to eukaryotic lipid rafts reveals a level of sophistication in signal transduction and membrane organization unexpected in bacteria. It opens new and promising venues to address intricate questions related to the functionality of lipid rafts by using bacteria as a more tractable system. This is the first report that uses bacteria as a working model to investigate a fundamental question that was previously raised while studying the role of eukaryotic lipid rafts. It also provides evidence of the critical role of these signaling platforms in orchestrating diverse physiological processes in prokaryotic cells.

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Role of lipid rafts in membrane delivery of renal epithelial Na+-K+-ATPase, thick ascending limb

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00166.2006 ◽

2007 ◽

Vol 292 (3) ◽

pp. R1328-R1337 ◽

Cited By ~ 24

Author(s):

Pia Welker ◽

Beate Geist ◽

Jan-Henning Frühauf ◽

Michele Salanova ◽

David A. Groneberg ◽

...

Keyword(s):

Lipid Rafts ◽

Rat Kidney ◽

Membrane Microdomains ◽

Cholesterol Depletion ◽

Thick Ascending Limb ◽

Low Density ◽

Α Subunit ◽

Density Fraction ◽

Short Term Exposure

Lipid rafts are cholesterol- and shingolipid-enriched membrane microdomains implicated in membrane signaling and trafficking. To assess renal epithelial raft functions through the characterization of their associated membrane proteins, we have isolated lipid rafts from rat kidney by sucrose gradient fractionation after detergent treatment. The low-density fraction was enriched in cholesterol, sphingolipid, and flotillin-1 known as lipid raft markers. Based on proteomic analysis of the low-density fraction, the protein with the highest significance score was the α-subunit of Na+-K+-ATPase (NKA), whose raft association was validated by simultaneous immunoblotting. The β-subunit of NKA was copurified from the low-density fraction. To test the role of lipid rafts in sorting and membrane delivery of renal-transporting epithelia, we have chosen to study thick ascending limb (TAL) epithelium for its high NKA activity and the property to be stimulated by antidiuretic hormone (ADH). Cultured rabbit TAL cells were studied. Cholesterol depletion and detergent extraction at warmth caused a shift of NKA to the higher-density fractions. Comparative preparations from blood monocytes revealed the absence of NKA from rafts in these nonpolarized cells. Short-term exposure of rabbit TAL cells to ADH (1 h) caused translocation and enhanced raft association of NKA via cAMP activation. Preceding cholesterol depletion prevented this effect. TAL-specific, glycosylphosphatidylinositol-anchored Tamm Horsfall protein was copurified with NKA in the same raft fraction, suggesting functional interference between these products. These results may have functional implications regarding the turnover, trafficking, and regulated surface expression of NKA as the major basolateral ion transporter of TAL.

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