scholarly journals Acid-sensing ion channel 3 (ASIC3) cell surface expression is modulated by PSD-95 within lipid rafts

2008 ◽  
Vol 295 (3) ◽  
pp. C732-C739 ◽  
Author(s):  
Jayasheel O. Eshcol ◽  
Anne Marie S. Harding ◽  
Tomonori Hattori ◽  
Vivian Costa ◽  
Michael J. Welsh ◽  
...  
Keyword(s):  
Cell Surface ◽  
Ion Channel ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Ph Sensor ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Membrane Microdomains ◽  
Metabolic Disturbances ◽  
Raft Fraction

Acid-sensing ion channel 3 (ASIC3) is a H+-gated cation channel primarily found in sensory neurons, where it may function as a pH sensor in response to metabolic disturbances or painful conditions. We previously found that ASIC3 interacts with the postsynaptic density protein PSD-95 through its COOH terminus, which leads to a decrease in ASIC3 cell surface expression and H+-gated current. PSD-95 has been implicated in recruiting proteins to lipid rafts, which are membrane microdomains rich in cholesterol and sphingolipids that organize receptor/signaling complexes. We found ASIC3 and PSD-95 coimmunoprecipitated within detergent-resistant membrane fractions. When cells were exposed to methyl-β-cyclodextrin to deplete membrane cholesterol and disrupt lipid rafts, PSD-95 localization to lipid raft fractions was abolished and no longer inhibited ASIC3 current. Likewise, mutation of two cysteine residues in PSD-95 that undergo palmitoylation (a lipid modification that targets PSD-95 to lipid rafts) prevented its inhibition of ASIC3 current and cell surface expression. In addition, we found that cell surface ASIC3 is enriched in the lipid raft fraction. These data suggest that PSD-95 and ASIC3 interact within lipid rafts and that this raft interaction is required for PSD-95 to modulate ASIC3.

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

Blood ◽  
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.

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

Blood ◽  
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.

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

2003 ◽  
Vol 77 (11) ◽  
pp. 6265-6273 ◽  
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.

scholarly journals Intracellular Retention of Glycosylphosphatidyl Inositol-Linked Proteins in Caveolin-Deficient Cells

2002 ◽  
Vol 22 (11) ◽  
pp. 3905-3926 ◽  
Author(s):  
Federica Sotgia ◽  
Babak Razani ◽  
Gloria Bonuccelli ◽  
William Schubert ◽  
Michela Battista ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Lipid Rafts ◽  
Recombinant Expression ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Null Mouse ◽  
Tissue Samples ◽  
Caveolin 1 ◽  
Glycosylphosphatidyl Inositol

ABSTRACT The relationship between glycosylphosphatidyl inositol (GPI)-linked proteins and caveolins remains controversial. Here, we derived fibroblasts from Cav-1 null mouse embryos to study the behavior of GPI-linked proteins in the absence of caveolins. These cells lack morphological caveolae, do not express caveolin-1, and show a ∼95% down-regulation in caveolin-2 expression; these cells also do not express caveolin-3, a muscle-specific caveolin family member. As such, these caveolin-deficient cells represent an ideal tool to study the role of caveolins in GPI-linked protein sorting. We show that in Cav-1 null cells GPI-linked proteins are preferentially retained in an intracellular compartment that we identify as the Golgi complex. This intracellular pool of GPI-linked proteins is not degraded and remains associated with intracellular lipid rafts as judged by its Triton insolubility. In contrast, GPI-linked proteins are transported to the plasma membrane in wild-type cells, as expected. Furthermore, recombinant expression of caveolin-1 or caveolin-3, but not caveolin-2, in Cav-1 null cells complements this phenotype and restores the cell surface expression of GPI-linked proteins. This is perhaps surprising, as GPI-linked proteins are confined to the exoplasmic leaflet of the membrane, while caveolins are cytoplasmically oriented membrane proteins. As caveolin-1 normally undergoes palmitoylation on three cysteine residues (133, 143, and 156), we speculated that palmitoylation might mechanistically couple caveolin-1 to GPI-linked proteins. In support of this hypothesis, we show that palmitoylation of caveolin-1 on residues 143 and 156, but not residue 133, is required to restore cell surface expression of GPI-linked proteins in this complementation assay. We also show that another lipid raft-associated protein, c-Src, is retained intracellularly in Cav-1 null cells. Thus, Golgi-associated caveolins and caveola-like vesicles could represent part of the transport machinery that is necessary for efficiently moving lipid rafts and their associated proteins from the trans-Golgi to the plasma membrane. In further support of these findings, GPI-linked proteins were also retained intracellularly in tissue samples derived from Cav-1 null mice (i.e., lung endothelial and renal epithelial cells) and Cav-3 null mice (skeletal muscle fibers).

scholarly journals Activity and PI3-kinase dependent trafficking of the intestinal anion exchanger downregulated in adenoma depend on its PDZ interaction and on lipid rafts

2010 ◽  
Vol 299 (4) ◽  
pp. G907-G920 ◽  
Author(s):  
S. Lissner ◽  
L. Nold ◽  
C.-J. Hsieh ◽  
J. R. Turner ◽  
M. Gregor ◽  
...  
Keyword(s):  
Cell Surface ◽  
Lipid Rafts ◽  
Adaptor Proteins ◽  
Surface Expression ◽  
Pi3 Kinase ◽  
Cell Surface Expression ◽  
Binding Motif ◽  
Wild Type ◽  
Pdz Proteins ◽  
Nonionic Detergent

The Cl/HCO3 exchanger downregulated in adenoma (DRA) mediates electroneutral NaCl absorption in the intestine together with the apical Na/H exchanger NHE3. Lipid rafts (LR) modulate transport activity and are involved in phosphatidylinositol 3-kinase (PI3-kinase)-dependent trafficking of NHE3. Although DRA and NHE3 interact via PDZ adaptor proteins of the NHERF family, the role of LR and PDZ proteins in the regulation of DRA is unknown. We examined the association of DRA with LR using the nonionic detergent Triton X-100. DRA cofractionated with LR independently of its PDZ binding motif. Furthermore, DRA interacts with PDZK1, E3KARP, and IKEPP in LR, although their localization within lipid rafts is independent of DRA. Disruption of LR integrity resulted in the disappearance of DRA from LR, in a decrease of its surface expression and in a reduction of its activity. In HEK cells the inhibition of DRA by LR disruption was entirely dependent on the presence of the PDZ interaction motif. In addition, in Caco-2/BBE cells the inhibition by LR disruption was more pronounced in wild-type DRA than in mutated DRA (DRA-ETKFminus; lacking the PDZ binding motif)-expressing cells. Inhibition of PI3-kinase decreased the activity and the cell surface expression of wild-type DRA but not of DRA-ETKFminus; the partitioning into LR was unaffected. Furthermore, simultaneous inhibition of PI3-kinase and disruption of LR did not further decrease DRA activity and cell surface expression compared with LR disruption only. These results suggest that the activity of DRA depends on its LR association, on its PDZ interaction, and on PI3-kinase activity.

scholarly journals Internalization of UT-A1 urea transporter is dynamin dependent and mediated by both caveolae- and clathrin-coated pit pathways

2010 ◽  
Vol 299 (6) ◽  
pp. F1389-F1395 ◽  
Author(s):  
Haidong Huang ◽  
Xiuyan Feng ◽  
Jieqiu Zhuang ◽  
Otto Fröhlich ◽  
Janet D. Klein ◽  
...  
Keyword(s):  
Cell Surface ◽  
Lipid Raft ◽  
Surface Expression ◽  
Membrane Lipid ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Protein Accumulation ◽  
Intracellular Loop ◽  
Cell Plasma ◽  
Cell Surface Biotinylation

Dynamin is a large GTPase involved in several distinct modes of cell endocytosis. In this study, we examined the possible role of dynamin in UT-A1 internalization. The direct relationship of UT-A1 and dynamin was identified by coimmunoprecipitation. UT-A1 has cytosolic NH2 and COOH termini and a large intracellular loop. Dynamin specifically binds to the intracellular loop of UT-A1, but not the NH2 and COOH termini. In cell surface biotinylation experiments, coexpression of dynamin and UT-A1 in HEK293 cells resulted in a decrease of UT-A1 cell surface expression. Conversely, cells expressing dynamin mutant K44A, which is deficient in GTP binding, showed an increased accumulation of UT-A1 protein on the cell surface. Cell plasma membrane lipid raft fractionation experiments revealed that blocking endocytosis with dynamin K44A causes UT-A1 protein accumulation in both the lipid raft and nonlipid raft pools, suggesting that both caveolae- and clathrin-mediated mechanisms may be involved in the internalization of UT-A1. This was further supported by 1) small interfering RNA to knock down either caveolin-1 or μ2 reduced UT-A1 internalization in HEK293 cells and 2) inhibition of either the caveolae pathway by methyl-β-cyclodextrin or the clathrin pathway by concanavalin A caused UT-A1 cell membrane accumulation. Functionally, overexpression of dynamin, caveolin, or μ2 decreased UT-A1 urea transport activity and decreased UT-A1 cell surface expression. We conclude that UT-A1 endocytosis is dynamin-dependent and mediated by both caveolae- and clathrin-coated pit pathways.

scholarly journals PSD-95 and SAP97 Exhibit Distinct Mechanisms for Regulating K+ Channel Surface Expression and Clustering

2000 ◽  
Vol 148 (1) ◽  
pp. 147-157 ◽  
Author(s):  
Amanda M. Tiffany ◽  
Louis N. Manganas ◽  
Eunjoon Kim ◽  
Yi-Ping Hsueh ◽  
Morgan Sheng ◽  
...  
Keyword(s):  
Cell Surface ◽  
Ion Channel ◽  
Membrane Vesicles ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
K Channel ◽  
Neuronal Membrane ◽  
K Channels ◽  
Kv Channels ◽  
Abundance And Distribution

Mechanisms of ion channel clustering by cytoplasmic membrane-associated guanylate kinases such as postsynaptic density 95 (PSD-95) and synapse-associated protein 97 (SAP97) are poorly understood. Here, we investigated the interaction of PSD-95 and SAP97 with voltage-gated or Kv K+ channels. Using Kv channels with different surface expression properties, we found that clustering by PSD-95 depended on channel cell surface expression. Moreover, PSD-95–induced clusters of Kv1 K+ channels were present on the cell surface. This was most dramatically demonstrated for Kv1.2 K+ channels, where surface expression and clustering by PSD-95 were coincidentally promoted by coexpression with cytoplasmic Kvβ subunits. Consistent with a mechanism of plasma membrane channel–PSD-95 binding, coexpression with PSD-95 did not affect the intrinsic surface expression characteristics of the different Kv channels. In contrast, the interaction of Kv1 channels with SAP97 was independent of Kv1 surface expression, occurred intracellularly, and prevented further biosynthetic trafficking of Kv1 channels. As such, SAP97 binding caused an intracellular accumulation of each Kv1 channel tested, through the accretion of SAP97 channel clusters in large (3–5 μm) ER-derived intracellular membrane vesicles. Together, these data show that ion channel clustering by PSD-95 and SAP97 occurs by distinct mechanisms, and suggests that these channel-clustering proteins may play diverse roles in regulating the abundance and distribution of channels at synapses and other neuronal membrane specializations.

N -glycan-dependent cell-surface expression of the P2Y 2 receptor and N -glycan-independent distribution to lipid rafts

2017 ◽  
Vol 485 (2) ◽  
pp. 427-431 ◽  
Author(s):  
Tetsuto Nakagawa ◽  
Chihiro Takahashi ◽  
Hitomi Matsuzaki ◽  
Shohei Takeyama ◽  
Shinpei Sato ◽  
...  
Keyword(s):  
Cell Surface ◽  
Lipid Rafts ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Dependent Cell ◽  
Independent Distribution
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