Tea polyphenol epigallocatechin-3-gallate associates with plasma membrane lipid rafts: lipid rafts mediate anti-allergic action of the catechin

High-affinity IgE receptor FcepsilonRI is key molecule in the IgE-mediated allergic reactions. Epigallocatechin-3-gallate (EGCG) has a suppressive effect of the expression of the FcepsilonRI. We show here that EGCG highly associates with plasma membrane microdomains, lipid rafts. The disruption of these lipid rafts caused a reduction of the amount of raft-associated EGCG and the FcepsilonRI -suppressive effect of EGCG. These results suggest that the interaction between EGCG and the lipid rafts is important for EGCG's ability to downregulate FcepsilonRI expression.     

A lipid raft-associated 67kDa laminin receptor mediates suppressive effect of epigallocatechin-3-O-gallate on FcepsilonRI expression

(-)-Epigallocatechin-3-O-gallate (EGCG), a major green tea polyphenol, has previously exhibited a suppressive effect on the expression of the high-affinity IgE receptor (FcepsilonRI). This effect has been shown to be elicited by interaction with the plasma membrane microdomain lipid rafts. Recently, we have identified the 67 kDa laminin receptor (67LR) as a cell surface EGCG receptor that mediates an anti-cancer action. Here we show that the 67LR is highly associated with lipid rafts on human basophilic KU812 cells. Experiments using 67LR-enhanced and -reduced cells revealed that the EGCG's ability to downregulate FcepsilonRI expression correlated with the amount of 67LR. Thus, these results suggest that the lipid raft-associated 67LR plays an important role in mediating the FcepsilonRI-suppressive action of EGCG.     

A difference between epigallocatechin-3-gallate and epicatechin-3-gallate on anti-allergic effect is dependent on their distinct distribution to lipid rafts

The high-affinity IgE receptor FcepsilonRI expresses on the cell surface of mast cells and basophils, which is the key molecule in allergic reactions. We previously found that the major green tea catechin, (-)-epigallocatechin-3-O-gallate (EGCG), has the suppressive effect of the FcepsilonRI expression in the human basophilic KU812 cells, whereas (-)-epicatechin-3-O-gallate (ECG) has not. For understanding the mechanism of catechins, interactions of catechins with cellular membranes were investigated. Both catechins were shown to bind the cell surface of KU812 cells by surface plasmon resonance assay. EGCG highly associated with cholesterol- and sphingolipid-enriched membrane microdomains known as lipid rafts. On the other hand, the level of ECG in rafts was lower than that of EGCG, suggesting that the association with lipid rafts may have an important role in the FcepsilonRI-suppressive effect of catechins.     

Lipid rafts mediate epigallocatechin-3-gallate- and green tea extract-dependent viability of human colon adenocarcinoma COLO 205 cells; clusterin affects lipid rafts-associated signaling pathways

Epigallocatechin-3-gallate (EGCG) is an important bioactive constituent of green tea extract (GTE) that was widely believed to reduce proliferation of many cancer cell lines. The purpose of this study was to verify the possible pro-apoptotic action of GTE/EGCG in human colon adenocarcinoma COLO 205 cells. The effect of EGCG/GTE treatments on cell viability was studied using methyl thiazolyl tetrazolium (MTT) assay. Cell proliferation was assessed with crystal violet staining, whereas protein expression levels were evaluated by western blotting followed by densitometric analysis. Obtained results were analyzed statistically. Surprisingly, EGCG/GTE dose-dependently up-regulated COLO 205 cells viability and proliferation. Observed effects were mediated by lipid rafts, as cholesterol depletion significantly prevented EGCG/GTE-dependent cell survival. Furthermore, treatment of COLO 205 cells with EGCG/GTE resulted in activation of MEK/ERK1/2, but not Akt1/2/GSK-3β signaling pathway. The presence of MEK inhibitor - PD98059 but not PI3-K inhibitor - LY294002, both reduced EGCG/GTE-induced ERK1/2 activation and the proliferative effect of catechins. Furthermore, EGCG/GTE stimulated secretory clusterin (sClu) expression level, which underwent complex control through lipid rafts/PKC/Wnt/β-catenin system. Our studies demonstrated that EGCG and GTE stimulate cell survival and proliferation of COLO 205 cells in a lipid rafts-dependent manner via at least MEK/ERK1/2 signaling pathway. Furthermore, EGCG/GTE mediated positive effects on viability and mitogenicity of COLO 205, while suppression of β-catenin activity was positively correlated with sClu clusterin expression.     

The involvement of the 67 kDa laminin receptor-mediated modulation of cytoskeleton in the degranulation inhibition induced by epigallocatechin-3-O-gallate

Recently, we have reported that (-)-epigallocatechin-3-O-gallate (EGCG) acts as an inhibitor of degranulation. However, the inhibitory mechanism for degranulation is still poorly understood. Here we show that suppression of exocytosis-related myosin II regulatory light chain phosphorylation and alteration of actin remodeling are involved in the inhibitory effect of EGCG on the calcium ionophore-induced degranulation from human basophilic KU812 cells. Surface plasmon resonance assay also revealed that EGCG binds to the cell surface, and the disruption of lipid rafts resulted in reduction of EGCG's ability. We have previously identified the raft-associated 67kDa laminin receptor (67LR) as an EGCG receptor on the cell surface. Treatment of the cells with anti-67LR antibody or RNA interference-mediated downregulation of 67LR expression abolished the effects of EGCG. These findings suggest that EGCG-induced inhibition of the degranulation includes the primary binding of EGCG to the cell surface 67LR and subsequent modulation of cytoskeleton.     

Phospholipid scramblase 1 modulates a selected set of IgE receptor-mediated mast cell responses through LAT-dependent pathway

Engagement of the IgE receptor (FcepsilonRI) on mast cells leads to the release of preformed and newly formed mediators as well as of cytokines. The signaling pathways responsible for these responses involve tyrosine phosphorylation of multiple proteins. We previously reported the phosphorylation on tyrosine of phospholipid scramblase 1 (PLSCR1) after FcepsilonRI aggregation. Here, PLSCR1 expression was knocked down in the RBL-2H3 mast cell line using short hairpin RNA. Knocking down PLSCR1 expression resulted in significantly impaired degranulation responses after FcepsilonRI aggregation and release of vascular endothelial growth factor, whereas release of MCP-1 was minimally affected. The release of neither leukotriene C4 nor prostaglandin D2 was altered by knocking down of PLSCR1. Analysis of FcepsilonRI-dependent signaling pathways revealed that whereas tyrosine phosphorylation of ERK and Akt was unaffected, tyrosine phosphorylation of LAT was significantly reduced in PLSCR1 knocked down cells. Tyrosine phosphorylation of phospholipase Cgamma1 and consequently the mobilization of calcium were also significantly reduced in these cells. In nonactivated mast cells, PLSCR1 was found in part in lipid rafts where it was further recruited after cell activation and was constitutively associated with Lyn and Syk but not with LAT or Fyn. Altogether, these data identify PLSCR1 as a novel amplifier of FcepsilonRI signaling that acts selectively on the Lyn-initiated LAT/phospholipase Cgamma1/calcium axis, resulting in potentiation of a selected set of mast cell responses.     

The high-affinity immunoglobulin-E receptor (FcepsilonRI) is endocytosed by an AP-2/clathrin-independent, dynamin-dependent mechanism

Aggregation of the high-affinity immunoglobulin E (IgE) receptor (FcepsilonRI), expressed on mast cells and basophils, initiates the immediate hypersensitivity reaction. Aggregated FcepsilonRI has been reported to rapidly migrate to lipid rafts in RBL-2H3 cells. We confirmed that aggregated FcepsilonRI is found in the lipid raft fractions of cellular lysates. Furthermore, we show that the cross-linked FcepsilonRI remains associated with detergent-resistant structures upon internalization. Previous morphological studies have reported that aggregated FepsiloncRI is endocytosed via clathrin-coated pits, which in general are not lipid raft associated. To address this apparent discrepancy, we employed siRNA to suppress expression of components of the clathrin-mediated internalization machinery, namely, clathrin heavy chain, and the AP-2 (alpha-adaptin or mu2-subunit). Transferrin receptor (TfR) is endocytosed by a clathrin-mediated process and, as expected, each transfected siRNA caused a two to threefold elevation of TfR surface expression and almost completely inhibited its endocytosis. In contrast, there was no effect on surface expression levels of FcepsilonRI nor on the endocytosis of the dinitrophenyl-human serum albumin (DNP-HSA)/IgE/FcepsilonRI complex. On the contrary, internalization of DNP-HSA/IgE/FcepsilonRI was inhibited by overexpression of a dominant-negative dynamin mutant. We conclude that internalization of cross-linked FcRI does not require the AP-2/clathrin complex but is dynamin-dependent and may be lipid raft mediated.     

Nerve growth factor stimulates the concentration of TrkA within lipid rafts and extracellular signal-regulated kinase activation through c-Cbl-associated protein

Nerve growth factor (NGF) acts through its receptor, TrkA, to elicit the neuronal differentiation of PC12 cells through the action of extracellular signal-regulated kinase 1 (ERK1) and ERK2. Upon NGF binding, TrkA translocates and concentrates in cholesterol-rich membrane microdomains or lipid rafts, facilitating formation of receptor-associated signaling complexes, activation of downstream signaling pathways, and internalization into endosomes. We have investigated the mechanisms responsible for the localization of TrkA within lipid rafts and its ability to activate ERK1 and ERK2. We report that NGF treatment results in the translocation of activated forms of TrkA to lipid rafts, and this localization is important for efficient activation of the ERKs. TrkA is recruited and retained within lipid rafts through its association with flotillin, an intrinsic constituent of these membrane microdomains, via the adapter protein, c-Cbl associated protein (CAP). Mutant forms of CAP that lack protein interaction domains block TrkA localization to lipid rafts and attenuate ERK activation. Importantly, suppression of endogenous CAP expression inhibited NGF-stimulated neurite outgrowth from primary dorsal root ganglion neurons. These data provide a mechanism for the lipid raft localization of TrkA and establish the importance of the CAP adaptor protein for NGF activation of the ERKs and neuronal differentiation.     

HB-EGF synthesis and release induced by cholesterol depletion of human epidermal keratinocytes is controlled by extracellular ATP and involves both p38 and ERK1/2 signaling pathways

The heparin-binding EGF-like growth factor (HB-EGF) is an autocrine/paracrine keratinocyte growth factor, which binds to the epidermal growth factor (EGF) receptor family and plays a critical role during the re-epithelialization of cutaneous wound by stimulating the keratinocytes proliferation and migration. In this study, cellular stressing condition in autocrine cultures of human keratinocytes was induced by cholesterol depletion using methyl-beta-cyclodextrin (MβCD). MβCD treatment induces the expression and the release of HB-EGF. By analysis of the culture media, large amounts of cellular ATP were measured particularly after 1 h of MβCD treatment. To investigate whether ATP contributes to the expression of HB-EGF, the nonhydrolyzable ATP analogue, ATP-γ-S, was used to mimic the extracellular ATP released. We report that keratinocytes stimulated with ATP-γ-S induce HB-EGF expression and activate EGFR and ERK1/2. Using an antagonist of P2 purinergic receptors, we demonstrate that HB-EGF synthesis induced by lipid rafts disruption is dependent on ATP interaction with P2 purinergic receptors. Moreover, our data suggest that both MAPKs p38 and ERK1/2 are involved together or independently in the regulation of HB-EGF gene expression. These findings provide new insight into the signaling pathway by which HB-EGF is expressed after lipid rafts disruption. In summary, after lipid raft disruption, keratinocytes release large amount of extracellular ATP. ATP induces HB-EGF synthesis and release by interacting with the P2 purinergic receptor and through p38 and ERK1/2 signaling in response to a challenging environment. A release of ATP acts as an early stress response in keratinocytes.     

Cutting Edge: Transmembrane phosphoprotein Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomains as a negative feedback regulator of mast cell signaling through the FcepsilonRI

Tyrosine phosphorylation in the cytoplasmic domains of FcepsilonRI by the Src family kinase Lyn initiates a signaling cascade leading to mast cell activation. In this study, we show that a recently identified transmembrane protein, Csk-binding protein (Cbp), also known as phospoprotein associated with glycosphingolipid-enriched microdomains (PAG), negatively regulates FcepsilonRI signaling. In rat basophilic leukemia (RBL)-2H3 cells, the levels of tyrosine phosphorylation of Cbp/PAG and its association with Csk, a negative regulator for Lyn, significantly elevate immediately after aggregation of FcepsilonRI. An overexpression of Cbp/PAG in RBL-2H3 cells inhibits FcepsilonRI-mediated cell activation. This is accompanied with decreased levels of tyrosine phosphorylation of FcepsilonRI, association of FcepsilonRI with Lyn, and FcepsilonRI-associated tyrosine kinase activity. These findings combined with the fact that Cbp/PAG, Lyn, and aggregated FcepsilonRI are localized to lipid rafts, suggest that upon FcepsilonRI aggregation Cbp/PAG down-regulates the receptor-associated Lyn activity through relocating Csk to rafts, thereby efficiently mediating feedback inhibition of FcepsilonRI signaling.     

Structure-Function Analysis of Lyn Kinase Association with Lipid Rafts and Initiation of Early Signaling Events after Fcɛ Receptor I Aggregation

The first step in immunoreceptor signaling is represented by ligand-dependent receptor aggregation, followed by receptor phosphorylation mediated by tyrosine kinases of the Src family. Recently, sphingolipid- and cholesterol-rich plasma membrane microdomains, called lipid rafts, have been identified and proposed to function as platforms where signal transduction molecules may interact with the aggregated immunoreceptors. Here we show that aggregation of the receptors with high affinity for immunoglobulin E (FcepsilonRI) in mast cells is accompanied by a co-redistribution of the Src family kinase Lyn. The co-redistribution requires Lyn dual fatty acylation, Src homology 2 (SH2) and/or SH3 domains, and Lyn kinase activity, in cis or in trans. Palmitoylation site-mutated Lyn, which is anchored to the plasma membrane but exhibits reduced sublocalization into lipid rafts, initiates the tyrosine phosphorylation of FcepsilonRI subunits, Syk protein tyrosine kinase, and the linker for activation of T cells, along with an increase in the concentration of intracellular Ca(2+). However, Lyn mutated in both the palmitoylation and myristoylation sites does not anchor to the plasma membrane and is incapable of initiating FcepsilonRI phosphorylation and early signaling events. These data, together with our finding that a constitutively tyrosine-phosphorylated FcepsilonRI does not exhibit an increased association with lipid rafts, suggest that FcepsilonRI phosphorylation and early activation events can be initiated outside of lipid rafts.     

Possible Protective Effect of Membrane Lipid Rafts against Interleukin-1β-Mediated Anti-Proliferative Effect in INS-1 Cells

We recently reported that pancreatic islets from pre-diabetic rats undergo an inflammatory process in which IL-1β takes part and controls β-cell function. In the present study, using the INS-1 rat pancreatic β-cell line, we investigated the potential involvement of membrane-associated cholesterol-enriched lipid rafts in IL-1β signaling and biological effects on insulin secretion, β-cell proliferation and apoptosis. We show that, INS-1 cells exposure to increasing concentrations of IL-1β leads to a progressive inhibition of insulin release, an increase in the number of apoptotic cells and a dose-dependent decrease in pancreatic β-cell proliferation. Disruption of membrane lipid rafts markedly reduced glucose-stimulated insulin secretion but did not affect either cell apoptosis or proliferation rate, demonstrating that membrane lipid raft integrity is essential for β-cell secretory function. In the same conditions, IL-1β treatment of INS-1 cells led to a slight further decrease in insulin secretion for low concentrations of the cytokine, and a more marked one, similar to that observed in normal cells for higher concentrations. These effects occurred together with an increase in iNOS expression and surprisingly with an upregulation of tryptophane hydroxylase and protein Kinase C in membrane lipid rafts suggesting that compensatory mechanisms develop to counteract IL-1β inhibitory effects. We also demonstrate that disruption of membrane lipid rafts did not prevent cytokine-induced cell death recorded after exposure to high IL-1β concentrations. Finally, concerning cell proliferation, we bring strong evidence that membrane lipid rafts exert a protective effect against IL-1β anti-proliferative effect, possibly mediated at least partly by modifications in ERK and PKB expression/activities. Our results 1) demonstrate that IL-1β deleterious effects do not require a cholesterol-dependent plasma membrane compartmentalization of IL-1R1 signaling and 2) confer to membrane lipid rafts integrity a possible protective function that deserves to be considered in the context of inflammation and especially T2D pathogenesis.     

Mast cell-specific gangliosides and FcepsilonRI follow the same endocytic pathway from lipid rafts in RBL-2H3 cells.

Recent studies have shown that, in mast cells, membrane microdomains rich in cholesterol and glycosphingolipids called lipid rafts play an important role in FcepsilonRI signaling. The present study demonstrates that, in RBL-2H3 cells following stimulation, the mast cell-specific gangliosides associated with FcepsilonRI are internalized from lipid rafts along with the receptor. When the cells are labeled with iodinated antibodies against the gangliosides or against FcepsilonRI and the cell components are then fractionated on Percoll density gradients, in stimulated cells the gangliosides are internalized with the same kinetics as FcepsilonRI and at 3 hr are present in the dense lysosome fraction. Using transmission electron microscopy, with antibody against the gangliosides conjugated to horseradish peroxidase and antibody against FcepsilonRI conjugated to colloidal gold, it was possible to demonstrate that the gangliosides and FcepsilonRI are internalized in the same coated vesicles. At 5 min, the gangliosides and FcepsilonRI can be identified in early endosomes and at 3 hr are found together in acid phosphatase-positive lysosomes. This study demonstrates that the mast cell-specific gangliosides are internalized from lipid rafts in the same vesicles and traffic intracellularly with the same kinetics as FcepsilonRI. This study contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.     

Epstein-Barr Virus LMP1 Modulates Lipid Raft Microdomains and the Vimentin Cytoskeleton for Signal Transduction and Transformation

The Epstein-Barr virus (EBV) is an important human pathogen that is associated with multiple cancers. The major oncoprotein of the virus, latent membrane protein 1 (LMP1), is essential for EBV B-cell immortalization and is sufficient to transform rodent fibroblasts. This viral transmembrane protein activates multiple cellular signaling pathways by engaging critical effector molecules and thus acts as a ligand-independent growth factor receptor. LMP1 is thought to signal from internal lipid raft containing membranes; however, the mechanisms through which these events occur remain largely unknown. Lipid rafts are microdomains within membranes that are rich in cholesterol and sphingolipids. Lipid rafts act as organization centers for biological processes, including signal transduction, protein trafficking, and pathogen entry and egress. In this study, the recruitment of key signaling components to lipid raft microdomains by LMP1 was analyzed. LMP1 increased the localization of phosphatidylinositol 3-kinase (PI3K) and its activated downstream target, Akt, to lipid rafts. In addition, mass spectrometry analyses identified elevated vimentin in rafts isolated from LMP1 expressing NPC cells. Disruption of lipid rafts through cholesterol depletion inhibited PI3K localization to membranes and decreased both Akt and ERK activation. Reduction of vimentin levels or disruption of its organization also decreased LMP1-mediated Akt and ERK activation and inhibited transformation of rodent fibroblasts. These findings indicate that LMP1 reorganizes membrane and cytoskeleton microdomains to modulate signal transduction.     

Cannabinoid 1 receptor-dependent transactivation of fibroblast growth factor receptor 1 emanates from lipid rafts and amplifies extracellular signal-regulated kinase 1/2 activation in embryonic cortical neurons

G-protein coupled receptors may mediate their effects on neuronal growth and differentiation through activation of extracellular signal-regulated kinases 1/2 (ERK1/2), often elicited by transactivation of growth factor receptor tyrosine kinases. This elaborate signaling process includes inducible formation and trafficking of multiprotein signaling complexes and is facilitated by pre-ordained membrane microdomains, in particular lipid rafts. In this study, we have uncovered novel signaling interactions of cannabinoid receptors with fibroblast growth factor receptors, which depended on lipid rafts and led to ERK1/2 activation in primary neurons derived from chick embryo telencephalon. More specifically, the cannabinoid 1 receptor (CB1R) agonist methanandamide induced tyrosine phosphorylation and transactivation of fibroblast growth factor receptor (FGFR)1 via Src and Fyn, which drove an amplification wave in ERK1/2 activation. Transactivation of FGFR1 was accompanied by the formation of a protein kinase C ε-dependent multiprotein complex that included CB1R, Fyn, Src, and FGFR1. Recruitment of molecules increased with time of exposure to methanandamide, suggesting that in addition to signaling it also served trafficking of receptors. Upon agonist stimulation we also detected a rapid incorporation of CB1R, as well as activated Src and Fyn, and FGFR1 in lipid rafts. Most importantly, lipid raft integrity was a pre-requisite for CB1R-dependent complex formation. Our data provide evidence that lipid rafts may organize CB1 receptor proximal signaling events, namely activation of Src and Fyn, and transactivation of FGFR1 towards activation of ERK1/2 and induction of neuronal differentiation.     

Lipid raft-dependent and -independent signaling through HLA-DR molecules

Lipid rafts are plasma membrane microdomains that are highly enriched in signaling molecules and that act as signal transduction platforms for many immune receptors. The involvement of these microdomains in HLA-DR-induced signaling is less well defined. We examined the constitutive presence of HLA-DR molecules in lipid rafts, their possible recruitment into these microdomains, and the role of these microdomains in HLA-DR-induced responses. We detected significant amounts of HLA-DR molecules in the lipid rafts of EBV(+) and EBV(-) B cell lines, monocytic cell lines, transfected HeLa cells, tonsillar B cells, and human monocytes. Localization of HLA-DR in these microdomains was unaffected by the deletion of the cytoplasmic domain of both the alpha and beta chains. Ligation of HLA-DR with a bivalent, but not a monovalent, ligand resulted in rapid tyrosine phosphorylation of many substrates, especially Lyn, and activation of ERK1/2 MAP kinase. However, the treatment failed to induce further recruitment of HLA-DR molecules into lipid rafts. The HLA-DR-induced signaling events were accompanied by the induction of cell-cell adhesion that could be inhibited by PTK and Lyn but not ERK1/2 inhibitors. Disruption of lipid rafts by methyl-beta-cyclodextrin (MbetaCD) resulted in the loss of membrane raft association with HLA-DR molecules, inhibition of HLA-DR-mediated protein tyrosine phosphorylation and cell-cell adhesion. MbetaCD did not affect the activation of ERK1/2, which was absent from lipid rafts. These results indicate that although all the HLA-DR-induced events studied are dependent on HLA-DR dimerization, some require the presence of HLA-DR molecules in lipid rafts, whereas others do not.     

Early activation of sphingosine kinase in mast cells and recruitment to FcepsilonRI are mediated by its interaction with Lyn kinase

Sphingosine kinase has been recognized as an essential signaling molecule that mediates the intracellular conversion of sphingosine to sphingosine-1-phosphate. In mast cells, induction of sphingosine kinase and generation of sphingosine-1-phosphate have been linked to the initial rise in Ca(2+), released from internal stores, and to degranulation. These events either precede or are concomitant with the activation of phospholipase C-gamma and the generation of inositol trisphosphate. Here we show that sphingosine kinase type 1 (SPHK1) interacts directly with the tyrosine kinase Lyn and that this interaction leads to the recruitment of this lipid kinase to the high-affinity receptor for immunoglobulin E (FcepsilonRI). The interaction of SPHK1 with Lyn caused enhanced lipid and tyrosine kinase activity. After FcepsilonRI triggering, enhanced sphingosine kinase activity was associated with FcepsilonRI in sphingolipid-enriched rafts of mast cells. Bone marrow-derived mast cells from Lyn(-/)(-) mice, compared to syngeneic wild-type cells, were defective in the initial induction of SPHK1 activity, and the defect was overcome by retroviral Lyn expression. These findings position the activation of SPHK1 as an FcepsilonRI proximal event.     

The lipid raft proteins flotillins/reggies interact with Galphaq and are involved in Gq-mediated p38 mitogen-activated protein kinase activation through tyrosine kinase

The heterotrimeric G protein alpha q subunit (Galphaq) mediates a variety of cell functions by activating the effector molecule phospholipase Cbeta. Galphaq activity is regulated by G protein betagamma subunits, G protein-coupled receptors, RGS proteins, and Ric-8. In this study, we identified the lipid raft resident proteins, flotillin-1/reggie-2 and flotillin-2/reggie-1, as Galphaq-binding proteins. The interactions of Galphaq and flotillins were independent of the nucleotide-binding state of Galphaq, and the N-terminal portion of flotillins was critical for the interaction. A short interfering RNA-mediated knockdown of flotillins, particularly flotillin-2, attenuated the UTP-induced activation of p38 mitogen-activated protein kinase (MAPK) but not that of ERK1/2. The activation of p38 MAPK was inhibited by the Src family tyrosine kinase inhibitor PP2 and the cholesterol-depleting agent methyl-beta-cyclodextrin, which is generally used for the disruption of lipid rafts. In contrast, the activation of ERK1/2 was not inhibited by these compounds. These lines of evidence suggested that a Gq-coupled receptor activates specifically p38 MAPK through lipid rafts and Src kinase activation, in which flotillins positively modulate the Gq signaling.