activity of EGFR by designing small molecules against the tyrosine kinase domain (erlotinib, gefitinib, and lapatinib) or antibodies against the ligand binding domains (cetuximab and panitumumab) [29,30]. Importantly, there is substantial experimental, epidemiological, and clinical evidence suggesting that consumption of n-3 polyunsaturated fatty acids (PUFA), including docosahexaenoic acid (DHA, 22:6D4,7,10,13,16,19) and eicosapentaenoic acid (EPA, 20:5D5,8,11,14,17) is protective against colon tumorigenesis [31,32,33,34,35,36,37,38,39,40,41,42,43,44,45]. However, the exact mechanisms by which n-3 PUFA function as chemopre- ventive agents have not been fully elucidated. Recent evidence suggests that perturbation of cell signaling events emanating from lipid rafts could be one mechanism of action of n-3 PUFA, specifically DHA [46,47,48]. DHA can profoundly influence cellular membrane composition and has been shown to have significant effects on plasma membrane properties, including membrane fluidity, phase behavior, permeability, fusion, flip-flop, and protein function following incorporation into membrane phospholipids [48,49]. Due to its high level of unsaturation, DHA has very poor affinity for cholesterol, which is enriched in lipid raft regions of the plasma membrane [50]. Studies conducted in various cell types have shown that treatment with DHA can alter the size of lipid rafts as well as signaling that is known to occur within rafts [47,51,52,53]. Furthermore, evidence suggests that treatment of cells with DHA results in exclusion of certain proteins from lipid rafts causing disruption of signal transduction events [46]. Therefore, we hypothesized that DHA exerts its effect on colon cancer in part by altering the localization and signaling of EGFR. Two initial studies have assessed the effects of n-3 PUFA on EGFR localization and signaling. Initially, a combination of EPA and DHA was shown to alter the localization of EGFR within the plasma membrane of a breast cancer cell line. The altered localization was concurrent with increased ligand-induced EGFR phosphorylation and downstream activation of p38MAPK [54]. The second study utilized DHA alone, and similarly reported an alteration in EGFR localization in lung and breast cancer cell lines [55]. Although EGFR phosphorylation and downstream signaling was only investigated under basal conditions, the increase in EGFR phosphorylation was associated with a suppression of Ras and ERK1/2 activation upon DHA treatment. This observation, although intriguing, did not directly address how n-3 PUFA impact EGFR functionality since receptor activation was never examined within the context of a ligand specific for the EGFR. Therefore, it is ambiguous whether the suppressive effects observed are due to alterations in EGFR or other parallel signaling pathways. Additionally, these studies only assessed a single downstream cascade from EGFR. Consequently, we set out to clarify the precise effect of n-3 PUFA on EGFR signaling in the colon. In this study, we demonstrate that treatment with DHA alters the localization of EGFR within the plasma membrane. Additionally, DHA induces an increase in ligand-induced EGFR phosphorylation with a paradoxical decrease in downstream signaling. These results reveal a novel mechanism by which DHA suppresses cell signaling by perturbing localization of key signaling mediators within lipid rafts. The suppressive effect on EGFR signaling is enhanced by DHA-induced increases in EGFR internalization and degradation. Furthermore, our data demon- strate an uncoupling of receptor phosphorylation from down- stream signaling that may affect our understanding of membrane localized receptor-mediated signaling events. Regulation of EGFR Signal Transduction Results DHA Reduces Partitioning of EGFR into Lipid Raft Domains Lateral organization of EGFR within the plasma membrane is directly linked to receptor function. Previous reports have shown that n-3 PUFA can alter the localization of EGFR within the plasma membrane of lung and breast cancer cells [54,55]. Therefore, we first determined the effect of DHA on localization of EGFR within the plasma membrane of colonocytes. Initial experiments were conducted using the immortalized Young Adult Mouse Colonocyte (YAMC) cell line, originally described by Whitehead and colleagues [56]. We employed two complemen- tary, established methods to investigate the localization of EGFR. First, a detergent-free method of plasma membrane fractionation to isolate lipid raft enriched domains was utilized [57]. We chose to use a detergent-free method due to strong evidence suggesting that detergent-based methods of extraction can lead to artifacts [58]. Cells were either untreated (control; no fatty acid) or incubated with 50 mM DHA or linoleic acid (LA, 18:2n26; control fatty acid) for 72 h. For the final 16–18 h of fatty acid treatment, cells were incubated in low serum media (0.5% FBS) prior to harvesting because EGFR has been shown to be localized to lipid rafts prior to activation. The bulk plasma membrane was isolated and fractionated on density gradients into three fractions, and each fraction was analyzed using Western blotting. The lowest density fraction from each treatment was enriched with caveolin and GM-1, which served as lipid raft markers, and depleted of clathrin, which is excluded from lipid rafts, indicating that this fraction is enriched with lipid rafts. In control and LA treated cells, the majority of EGFR was concentrated in the lipid raft enriched fraction of the plasma membrane (Figure 1A). However, in DHA treated cells, EGFR displayed a more even distribution across all plasma membrane fractions, indicating a lateral reorganization of EGFR. Additionally, confocal fluorescence microscopy was used to determine localization of EGFR. Control and fatty acid treated cells were co-transfected with monomeric GFP-tagged EGFR (EGFR-mGFP) and RFP-tagged truncated H-Ras (RFP-tH). RFP- tH, which is targeted by a CAAX motif that is both palmitoylated and farnesylated, is a well-established lipid raft marker that is localized exclusively to low density fractions on sucrose gradients [59,60]. In control and LA treated cells, plasma membrane EGFR-mGFP strongly colocalized with RFP-tH (Figure 1B). This colocalization was significantly decreased by treatment with DHA. Interestingly, in many of the DHA treated cells, EGFR-mGFP and RFP-tH formed patches that were localized in close proximity, but not overlapping. The overall localization of the lipid raft marker, RFP-tH, was altered by treatment with DHA, which is in agreement with a plethora of data suggesting that DHA alters lipid rafts [51–53]. Additionally, a large portion of EGFR in the DHA-treated cells was localized intracellularly. These comple- mentary datasets indicate that DHA treatment altered the plasma membrane organization of EGFR within colonocytes, which is consistent with previous observations in breast and lung cells [54,55]. DHA Suppresses EGFR Signaling but Increases EGFR Phosphorylation Due to the role that lipid rafts play in the regulation of EGFR activation and downstream signaling, we next investigated the effects of DHA on EGFR activation status and signaling in YAMC cells. Untreated and fatty acid treated cells were incubated in low serum media (0.5% FBS) overnight in order to reduce signaling from growth factors within the serum. PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e39682