Overexpression of epidermal growth factor receptor in NIH-3T3- transfected cells slows its lateral diffusion and rate of endocytosis

Interactions between membrane proteins are believed to be important for the induction of transmembrane signaling. Endocytosis is one of the responses which is regulated by both intracellular and extracellular signals. To study such interactions, we have measured the lateral mobility and rate of endocytosis of epidermal growth factor receptor in three transfected NIH-3T3 cell lines (HER84, HER22, and HER82) expressing 2 X 10(4), 2 X 10(5) and 1.5 X 10(6) EGF-receptors per cell, respectively. Using rhodamine-labeled EGF (Rh-EGF) and rhodamine-labeled monoclonal anti-EGF-receptor antibody (Rh-mAb-108), we measured twofold decreases in the lateral diffusion coefficients for each approximately 10-fold increase in EGF-receptor concentration. Since steric effects cannot account for such dependence, we propose that protein mobility within the membrane, which is determined by the rate of motion between immobile barriers, decreases due to aggregate formation. The rate of endocytosis also decreases twofold between the HER84 (2 X 10(4) receptors/cell) and HER22 (2 X 10(5) receptors/cell) cell lines, suggesting that it is diffusion limited. The comparable rates of endocytosis of the HER82 and HER22 cell lines suggest that at high receptor density endocytosis may be limited by the total number of sites for receptors in coated-pits and by their rate of recycling.     

N-cadherin association with lipid rafts regulates its dynamic assembly at cell-cell junctions in C2C12 myoblasts

Cadherins are homophilic cell-cell adhesion molecules implicated in cell growth, differentiation, and organization into tissues during embryonic development. They accumulate at cell-cell contact sites and act as adhesion-activated signaling receptors. Here, we show that the dynamic assembly of N-cadherin at cell-cell contacts involves lipid rafts. In C2C12 myoblasts, immunofluorescence and biochemical experiments demonstrate that N-cadherin present at cell-cell contacts is colocalized with lipid rafts. Disruption of lipid rafts leads to the inhibition of cell-cell adhesion and disorganization of N-cadherin-dependent cell-cell contacts without modifying the association of N-cadherin with catenins and its availability at the plasma membrane. Fluorescent recovery after photobleaching experiments demonstrate that at the dorsal plasma membrane, lipid rafts are not directly involved in the diffusional mobility of N-cadherin. In contrast, at cell-cell junctions N-cadherin association with lipid rafts allows its stabilization enabling the formation of a functional adhesive complex. We show that lipid rafts, as homophilic interaction and F-actin association, stabilize cadherin-dependent adhesive complexes. Homophilic interactions and F-actin association of N-cadherin are both required for its association to lipid rafts. We thus identify lipid rafts as new regulators of cadherin-mediated cell adhesion.     

Relationships between EGFR signaling-competent and endocytosis-competent membrane microdomains

Membrane microdomains, the so-called lipid rafts, function as platforms to concentrate receptors and assemble the signal transduction machinery. Internalization, in most cases, is carried out by different specialized structures, the clathrin-coated pits. Here, we show that several endocytic proteins are efficiently recruited to morphologically identified plasma membrane lipid rafts, upon activation of the epidermal growth factor (EGF) receptor (EGFR), a receptor tyrosine kinase. Analysis of detergent-resistant membrane fractions revealed that the EGF-dependent association of endocytic proteins with rafts is as efficient as that of signaling effector molecules, such as Grb2 or Shc. Finally, the EGFR, but not the nonsignaling transferrin receptor, could be localized in nascent coated pits that almost invariably contained raft membranes. Thus, specialized membrane microdomains have the ability to assemble both the molecular machineries necessary for intracellular propagation of EGFR effector signals and for receptor internalization.     

Sensitization of epidermal growth factor-induced signaling by bradykinin is mediated by c-Src. Implications for a role of lipid microdomains

Communication between receptor tyrosine kinase (RTK)- and G protein-coupled receptor (GPCR)-mediated signaling systems has received increasing attention in recent years. Here, we report that activation of G protein-coupled bradykinin B2 receptor induces an up-regulation of cellular responses mediated by epidermal growth factor receptor (EGFR) and provide essential mechanistic characteristics of this sensitization process. EGF, which failed to evoke detectable amount of calcium increase and neurotransmitter release when administrated alone in primary cultures of rat adrenal chromaffin cells and PC12 cells, became capable of inducing these responses specifically after bradykinin pretreatment. Both EGFR and non-receptor tyrosine kinase p60Src, whose kinase activities were required in the sensitization, were found to be enriched in cholesterol-rich lipid rafts. Bradykinin caused activation of p60Src and Src-dependent phosphorylation of the EGFR on Tyr-845 in lipid rafts, as well as recruitment of phospholipase C (PLC) gamma1 to the rafts. Depletion of cholesterol by methyl-beta-cyclodextrin disrupted the raft localization of EGFR and Src, as well as bradykinin-induced translocation of PLCgamma1. Furthermore, sensitization, which was impaired by cholesterol depletion, was restored by repletion of cholesterol. Therefore, we suggest that lipid rafts are essential participants in the regulation of receptor-mediated signal transduction and cross-talk via organizing signaling complexes in membrane microdomains.     

Lipid segregation and IgE receptor signaling: a decade of progress

Recent work to characterize the roles of lipid segregation in IgE receptor signaling has revealed a mechanism by which segregation of liquid ordered regions from disordered regions of the plasma membrane results in protection of the Src family kinase Lyn from inactivating dephosphorylation by a transmembrane tyrosine phosphatase. Antigen-mediated crosslinking of IgE receptors drives their association with the liquid ordered regions, commonly called lipid rafts, and this facilitates receptor phosphorylation by active Lyn in the raft environment. Previous work showed that the membrane skeleton coupled to F-actin regulates stimulated receptor phosphorylation and downstream signaling processes, and more recent work implicates cytoskeletal interactions with ordered lipid rafts in this regulation. These and other results provide an emerging view of the complex role of membrane structure in orchestrating signal transduction mediated by immune and other cell surface receptors.     

Constitutive localization of the gonadotropin-releasing hormone (GnRH) receptor to low density membrane microdomains is necessary for GnRH signaling to ERK

Specialized membrane microdomains known as lipid rafts are thought to contribute to G-protein coupled receptor (GPCR) signaling by organizing receptors and their cognate signaling molecules into discrete membrane domains. To determine if the GnRHR, an unusual member of the GPCR superfamily, partitions into lipid rafts, homogenates of alpha T3-1 cells expressing endogenous GnRHR or Chinese hamster ovary cells expressing an epitope-tagged GnRHR were fractionated through a sucrose gradient. We found the GnRHR and c-raf kinase constitutively localized to low density fractions independent of hormone treatment. Partitioning of c-raf kinase into lipid rafts was also observed in whole mouse pituitary glands. Consistent with GnRH induced phosphorylation and activation of c-raf kinase, GnRH treatment led to a decrease in the apparent electrophoretic mobility of c-raf kinase that partitioned into lipid rafts compared with unstimulated cells. Cholesterol depletion of alpha T3-1 cells using methyl-beta-cyclodextrin disrupted GnRHR but not c-raf kinase association with rafts and shifted the receptor into higher density fractions. Cholesterol depletion also significantly attenuated GnRH but not phorbol ester-mediated activation of extracellular signal-related kinase (ERK) and c-fos gene induction. Raft localization and GnRHR signaling to ERK and c-Fos were rescued upon repletion of membrane cholesterol. Thus, the organization of the GnRHR into low density membrane microdomains appears critical in mediating GnRH induced intracellular signaling.     

Membrane cholesterol depletion in cortical neurons highlights altered NMDA receptor functionality in a mouse model of amyotrophic lateral sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a chronic neurodegenerative disease affecting upper and lower motor neurons, with unknown aetiology. Lipid rafts, cholesterol enriched microdomains of the plasma membrane, have been linked to neurodegenerative disorders like ALS. The NMDA-receptor subcellular localization in lipid rafts is known to play many roles, from modulating memory strength to neurotoxicity. In this study, performed on the widely used G93A mouse model of ALS, we have shown an equal content of total membrane cholesterol in Control and G93A cortical cultures. Moreover, by electrophysiological studies, we have recorded NMDA- and AMPA-evoked currents which were not significantly different between the two neuronal populations. To study the role of membrane cholesterol on glutamate receptor functionality, we have analysed NMDA and AMPA receptors following cholesterol membrane depletion by methyl-β-cyclodextrin (MβCD). Interestingly, MβCD chronic treatment has provoked a significant reduction of NMDA-evoked currents in both cellular populations which was dose- and time-dependent but significantly higher in ALS neurons compared to Control. The different MβCD effect on NMDA-evoked currents was not due to a different membrane receptor subunit composition but seemed to cause in both neuronal populations a NMDA receptor membrane redistribution. MβCD treatment effect was receptor-specific since no alterations in the two neuronal populations were detected on AMPA receptors.These results lead us to speculate for an altered proteomic composition of lipid rafts in cortical mutated neurons and suggest the need for further studies on the lipid rafts composition and on their interaction with membrane receptors in ALS cortices.     

Parsing ERK activation reveals quantitatively equivalent contributions from epidermal growth factor receptor and HER2 in human mammary epithelial cells

HER2, a member of the epidermal growth factor receptor (EGFR) tyrosine kinase family, functions as an accessory EGFR signaling component and alters EGFR trafficking by heterodimerization. HER2 overexpression leads to aberrant cell behavior including enhanced proliferation and motility. Here we applied a combination of computational modeling and quantitative experimental studies of the dynamic interactions between EGFR and HER2 and their downstream activation of ERK to understand this complex signaling system. Using cells expressing different levels of HER2 relative to the EGFR, we could separate relative contributions of EGFR and HER2 to signaling amplitude and duration. Based on our model calculations, we demonstrated that, in contrast with previous suggestions in the literature, the intrinsic capabilities of EGFR and HER2 to activate ERK were quantitatively equivalent. We found that HER2-mediated effects on EGFR dimerization and trafficking were sufficient to explain the observed HER2-mediated amplification of epidermal growth factor-induced ERK signaling. Our model suggests that transient amplification of ERK activity by HER2 arises predominantly from the 2-to-1 stoichiometry of receptor kinase to bound ligand in EGFR/HER2 heterodimers compared with the 1-to-1 stoichiometry of the EGFR homodimer, but alterations in receptor trafficking yielding increased EGFR sparing cause the sustained HER2-mediated enhancement of ERK signaling.     

Observing the confinement potential of bacterial pore-forming toxin receptors inside rafts with nonblinking Eu(3+)-doped oxide nanoparticles

We track single toxin receptors on the apical cell membrane of MDCK cells with Eu-doped oxide nanoparticles coupled to two toxins of the pore-forming toxin family: α-toxin of Clostridium septicum and ε-toxin of Clostridium perfringens. These nonblinking and photostable labels do not perturb the motion of the toxin receptors and yield long uninterrupted trajectories with mean localization precision of 30 nm for acquisition times of 51.3 ms. We were thus able to study the toxin-cell interaction at the single-molecule level. Toxins bind to receptors that are confined within zones of mean area 0.40 ± 0.05 μm(2). Assuming that the receptors move according to the Langevin equation of motion and using Bayesian inference, we determined mean diffusion coefficients of 0.16 ± 0.01 μm(2)/s for both toxin receptors. Moreover, application of this approach revealed a force field within the domain generated by a springlike confining potential. Both toxin receptors were found to experience forces characterized by a mean spring constant of 0.30 ± 0.03 pN/μm at 37°C. Furthermore, both toxin receptors showed similar distributions of diffusion coefficient, domain area, and spring constant. Control experiments before and after incubation with cholesterol oxidase and sphingomyelinase show that these two enzymes disrupt the confinement domains and lead to quasi-free motion of the toxin receptors. Our control data showing cholesterol and sphingomyelin dependence as well as independence of actin depolymerization and microtubule disruption lead us to attribute the confinement of both receptors to lipid rafts. These toxins require oligomerization to develop their toxic activity. The confined nature of the toxin receptors leads to a local enhancement of the toxin monomer concentration and may thus explain the virulence of this toxin family.     

Human epidermal growth factor receptor (HER1) aligned on the plasma membrane adopts key features of Drosophila EGFR asymmetry

Current models suggest that ligand-binding heterogeneity in HER1 [human EGFR (epidermal growth factor receptor] arises from negative co-operativity in signalling HER1 dimers, for which the asymmetry of the extracellular region of the Drosophila EGFR has recently provided a structural basis. However, no asymmetry is apparent in the current crystal structure of the isolated extracellular region of HER1. This receptor also differs from the Drosophila EGFR in that negative co-operativity is found only in full-length receptors in cells. Structural insights into HER1 in epithelial cells, derived from FLIM (fluorescence lifetime imaging microscopy) and two-dimensional FRET (F?rster resonance energy transfer) combined with Monte Carlo and molecular dynamics simulations, have demonstrated a high-affinity ligand-binding HER1 conformation consistent with the extracellular region aligned flat on the plasma membrane. This conformation shares key features with that of the Drosophila EGFR, suggesting that the structural basis for negative co-operativity is conserved from invertebrates to humans, but that, in HER1, the extracellular region asymmetry requires interactions with the plasma membrane.     

Lipid raft localization of EGFR alters the response of cancer cells to the EGFR tyrosine kinase inhibitor gefitinib

Epidermal growth factor receptor (EGFR) is overexpressed in many cancer types including ~30% of breast cancers. Several small molecule tyrosine kinase inhibitors (TKIs) targeting EGFR have shown clinical efficacy in lung and colon cancers, but no benefit has been noted in breast cancer. Thirteen EGFR expressing breast cancer cell lines were analyzed for response to EGFR TKIs. Seven were found to be EGFR TKI resistant; while shRNA knockdown of EGFR determined that four of these cell lines retained the requirement of EGFR protein expression for growth. Interestingly, EGFR localized to plasma membrane lipid rafts in all four of these EGFR TKI-resistant cell lines, as determined by biochemical raft isolation and immunofluorescence. When lipid rafts were depleted of cholesterol using lovastatin, all four cell lines were sensitized to EGFR TKIs. In fact, the effects of the cholesterol biosynthesis inhibitors and gefitinib were synergistic. While gefitinib effectively abrogated phosphorylation of Akt- and mitogen-activated protein kinase in an EGFR TKI-sensitive cell line, phosphorylation of Akt persisted in two EGFR TKI-resistant cell lines, however, this phosphorylation was abrogated by lovastatin treatment. Thus, we have shown that lipid raft localization of EGFR correlates with resistance to EGFR TKI-induced growth inhibition and pharmacological depletion of cholesterol from lipid rafts decreases this resistance in breast cancer cell lines. Furthermore, we have presented evidence to suggest that when EGFR localizes to lipid rafts, these rafts provide a platform to facilitate activation of Akt signaling in the absence of EGFR kinase activity.     

Second cysteine-rich region of epidermal growth factor receptor contains targeting information for caveolae/rafts

Previous studies have shown that approximately 60% of the epidermal growth factor receptors (EGFRs) in quiescent fibroblasts are concentrated in the caveolae/raft fraction from purified plasma membranes. This high degree of localization suggests the EGFR contains targeting information for lipid domains. We have used mutagenesis to determine that the region of the receptor that controls targeting to caveolae/rafts resides in the juxtamembrane, second cysteine-rich region. A 60-amino acid-long sequence within this region that is continuous with the transmembrane domain was sufficient to target the transmembrane and cytoplasmic tails of both EGFR and the low density lipoprotein receptor to caveolae/rafts. Two N-linked sugars in this segment were not required for proper targeting, although unglycosylated wild-type receptors did not localize properly. We conclude that, in contrast to signals for coated pit localization that are in the cytoplasmic tail, the targeting information for caveolae/rafts is on the extracellular side of the EGFR very close to the membrane.     

NrCAM coupling to the cytoskeleton depends on multiple protein domains and partitioning into lipid rafts

NrCAM is a cell adhesion molecule of the L1 family that is implicated in the control of axonal growth. Adhesive contacts may promote advance of the growth cone by triggering the coupling of membrane receptors with the F-actin retrograde flow. We sought to understand the mechanisms leading to clutching the F-actin at the site of ligand-mediated clustering of NrCAM. Using optical tweezers and single particle tracking of beads coated with the ligand TAG-1, we analyzed the mobility of NrCAM-deletion mutants transfected in a neuroblastoma cell line. Deletion of the cytoplasmic tail did not prevent the coupling of NrCAM to the actin flow. An additional deletion of the FNIII domains to remove cis-interactions, was necessary to abolish the rearward movement of TAG-1 beads, which instead switched to a stationary behavior. Next, we showed that the actin-dependent retrograde movement of NrCAM required partitioning into lipid rafts as indicated by cholesterol depletion experiments using methyl-beta-cyclodextrin. Recruitment of the raft component caveolin-1 was induced at the adhesive contact between the cell surface and TAG-1 beads, indicating that enlarged rafts were generated. Photobleaching experiments showed that the lateral mobility of NrCAM increased with raft dispersion in these contact areas, further suggesting that TAG-1-coated beads induced the coalescence of lipid rafts. In conclusion, we propose that anchoring of NrCAM with the retrograde actin flow can be triggered by adhesive contacts via cooperative processes including interactions with the cytoplasmic tail, formation of cis-complex via the FNIII repeats, and lipid raft aggregation.     

Compartmentalization of EGFR in cellular membranes: Role of membrane rafts

There is now abundant evidence that the intracellular concentration of the EGFR and many other receptors for peptide hormones and growth factors is important for the temporal and spatial regulation of cell signaling. Spatial control is achieved by the selective compartmentalization of signaling components into endosomes. However further control may be effected by sequestration into sub‐domains within a given organelle such as membrane rafts which are dynamic, nano scale structures rich in cholesterol and sphingolipids. Current data suggest the presence of EGFRs in non‐caveolae membrane rafts. High doses of EGF seem to promote the sorting of EGFR to late endosomes through a raft/cholesterol dependant mechanism, implicating them in EGFR degradation. However our work and that of others has led us to propose a model in which membrane rafts in late endosomes sequester highly active EGFR leading to the recruitment and activation of MAPK in this compartment. J. Cell. Biochem. 109: 1103–1108, 2010. © 2010 Wiley‐Liss, Inc.     

Quantitative analysis of HER2-mediated effects on HER2 and epidermal growth factor receptor endocytosis: distribution of homo- and heterodimers depends on relative HER2 levels

Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR) family. Many cell types express multiple EGFR family members (including EGFR, HER2, HER3, and/or HER4) that interact to form an array of homo- and heterodimers. Differential trafficking of these receptors should strongly affect signaling through this system by changing substrate access and heterodimerization efficiency. Because of the complexity of these dynamic processes, we used a quantitative and computational model to understand their integrated operation. Parameters characterizing EGFR and HER2 interactions were determined using experimental data obtained from mammary epithelial cells constructed to express different levels of HER2, enabling us to estimate receptor-specific internalization rate constants and dimer uncoupling rate constants. Significant novel results obtained from this work are as follows: first, that EGFR homodimerization and EGFR/HER2 heterodimerization occur with comparable affinities; second, that EGFR/HER2 heterodimers traffic as single entities. Furthermore, model predictions of the relationship of HER2 expression levels to consequent distribution of EGFR homodimers and EGFR/HER2 heterodimers suggest that the levels of HER2 found on normal cells are barely at the threshold necessary to drive efficient heterodimerization. Thus, altering HER2 concentrations, either overall or local, could provide an effective mechanism for regulating EGFR/HER2 heterodimerization and may explain why HER2 overexpression found in some cancers has such a profound effect on cell physiology.     

Localization of receptors in lipid rafts can inhibit signal transduction

Processes of cell survival, division, differentiation, and death are guided by the binding of signal molecules to receptors, which activates intracellular signaling networks and ultimately elicits genetic, biochemical, or biomechanical responses within the cell. While intracellular mechanisms for these processes have been well studied, little attention has been given to the role extracellular ligand transport and binding may play in signal initiation. Recent studies have found that the localization of receptors in lipid rafts is critical for the functions of many signaling pathways. By concentrating membrane components, rafts may promote essential interactions for signaling. Lipid rafts can also have negative effects on signaling, but mechanisms remain elusive. We propose that raft-mediated receptor clustering can reduce signaling by prolonging the diffusion of ligands to their receptors. We quantify this effect using a simple diffusion-limited binding model that accounts for the spatial distribution of lipid rafts and receptors on the cell surface. We find that receptor clustering can reduce the apparent rate of receptor binding by up to 80%, consistent with observed increases in epidermal growth factor (EGF) binding by up to 100% following disruption of lipid rafts (Pike and Casey 2002 Biochemistry 41:10315-10322; Roepstorff et al. 2002 J Biol Chem 277:18954-18960). Failure to account for the effects of receptor clustering on rates of ligand binding can skew the interpretation of current methods of cancer diagnosis and treatment. Finally, we discuss how the activation of particular signaling pathways can change over time, depending, in part, on the overall level and spatial distribution of the receptors.     

Effects of cholesterol manipulation on the signaling of the human oxytocin receptor

We have recently shown that oxytocin inhibits cell growth when the vast majority of oxytocin receptors (OTRs) are excluded from detergent-resistant membranes (DRMs; the biochemical counterpart of lipid rafts), but has a strong mitogenic effect when the receptors are targeted to these plasma membrane domains upon fusion with caveolin-2, a resident raft protein. The aim of this study was to investigate whether the manipulation of total cell cholesterol can influence OTR localization and signaling. Our data indicate that cholesterol depletion in HEK-293 cells does not affect the signaling events mediated by the OTRs located outside DRMs. When treated with 2 mM methyl-beta-cyclodextrin (MbetaCD), the receptors remained outside and continued to inhibit cell growth. On the contrary, the MbetaCD treatment of cells expressing receptors fused to caveolin-2 led to their redistribution outside DRMs, and converted the receptor-mediated proliferative effect into cell growth inhibition. These data indicate that 1) once released from DRMs, the receptors fused to caveolin-2 signal exactly as wild-type OTRs and 2) their DRM location is responsible for the specific OTR signaling leading to cell proliferation. Finally, we evaluated whether cholesterol loading could force the OTRs into lipid rafts and change their signaling, but, after cell treatment with an MbetaCD/cholesterol complex, receptor stimulation continued to lead to cell growth inhibition, thus indicating that increasing cell cholesterol levels is not sufficient per se to affect OTR signaling.     

Unraveling the impact of lipid domains on the dimerization processes of single-molecule EGFRs of live cells

Epidermal growth factor receptor (EGFR/ErbB1) is a transmembrane protein that can drive cell growth and survival via the ligand-induced dimerization of receptors. Because dimerization is a common mechanism for signal transduction, it is important to improve our understanding of how the dimerization process and membrane structure regulate signal transduction. In this study, we examined the effect of lipid nanodomains on the dimerization process of EGFR molecules. We discovered that after ligand binding, EGFR molecules may move into lipid nanodomains. The lipid nanodomains surrounding two liganded EGFRs can merge during their correlated motion. The transition rates between different diffusion states of liganded EGFR molecules are regulated by the lipid domains. Our method successfully captures both the sensitivity of single-molecule processes and statistic accuracy of data analysis, providing insight into the connection between the mobile clustering process of receptors and the hierarchical structure of plasma membrane.     

Role of lipid rafts in E-cadherin-- and HGF-R/Met--mediated entry of Listeria monocytogenes into host cells

Listeria monocytogenes uptake by nonphagocytic cells is promoted by the bacterial invasion proteins internalin and InlB, which bind to their host receptors E-cadherin and hepatocyte growth factor receptor (HGF-R)/Met, respectively. Here, we present evidence that plasma membrane organization in lipid domains is critical for Listeria uptake. Cholesterol depletion by methyl-beta-cyclodextrin reversibly inhibited Listeria entry. Lipid raft markers, such as glycosylphosphatidylinositol-linked proteins, a myristoylated and palmitoylated peptide and the ganglioside GM1 were recruited at the bacterial entry site. We analyzed which molecular events require membrane cholesterol and found that the presence of E-cadherin in lipid domains was necessary for initial interaction with internalin to promote bacterial entry. In contrast, the initial interaction of InlB with HGF-R did not require membrane cholesterol, whereas downstream signaling leading to F-actin polymerization was cholesterol dependent. Our work, in addition to documenting for the first time the role of lipid rafts in Listeria entry, provides the first evidence that E-cadherin and HGF-R require lipid domain integrity for their full activity.     

Acid sphingomyelinase mediates 50-Hz magnetic field-induced EGF receptor clustering on lipid raft

Previously, we found that exposure to a 50-Hz magnetic field (MF) could induce epidermal growth factor receptor (EGFR) clustering and phosphorylation on cell surface. In order to explore the possible mechanisms, the roles of acid sphingomyelinase (ASMase) and lipid raft in MF-induced EGFR clustering were investigated in the present study. Human amnion epithelial (FL) cells were exposed to a 50-Hz MF at 0.4?mT for different durations. Intracellular ASMase activity was detected using the Amplex® Red Sphingomyelinase Assay Kit. EGFR clustering, ASMase, and lipid rafts on cell membrane were analyzed using confocal microscopy after indirect immunofluorescence staining. Results showed that disturbing lipid rafts with nystatin could inhibit MF-induced EGFR clustering, indicating that it was dependent on intact lipid raft. Exposure of FL cells to MF significantly enhanced ASMase activity and induced ASMase translocation to membrane that co-localized with lipid rafts. Treatment with imipramine, an ASMase inhibitor, inhibited the MF-induced EGFR clustering. This inhibitory effect could be blocked by the addition of C2-ceramide in the culture medium. It suggested that ASMase mediated the 50-Hz MF-induced EGFR clustering via ceramide which was produced from hydrolyzation on lipid rafts.