AbstractThe TNFR, TNF-R1, is localized to lipid raft and nonraft regions of the plasma membrane. Ligand binding sets in motion signaling cascades that promote the activation of p42mapk/erk2 and NF-κB. However, the role of receptor localization in the activation of downstream signaling events is poorly understood. In this study, we investigated the dynamics of TNF-R1 localization to lipid rafts and the consequences of raft localization on the activation of p42mapk/erk2 and NF-κB in primary cultures of mouse macrophages. Using sucrose density gradient ultracentrifugation and a sensitive ELISA to detect TNF-R1, we show that TNF-R1 is rapidly and transiently recruited to lipid rafts in response to TNF-α. Disruption of lipid rafts by cholesterol depletion prevented the TNF-α-dependent recruitment of TNF-R1 to lipid rafts and inhibited the activation of p42mapk/erk2, while the activation of NF-κB was unaffected. In addition, phosphorylated p42mapk/erk2, but not receptor interacting protein, I-κB kinase-γ, or I-κBα was detected in raft-containing fractions following TNF-α stimulation. These findings suggest that TNF-R1 is localized to both lipid raft and nonraft regions of the plasma membrane and that each compartment is capable of initiating different signaling responses. We propose that segregation of TNF-R1 to raft and nonraft regions of the plasma membrane contributes to the diversity of signaling responses initiated by TNF-R1.