TNFR1 contains a membrane-proximal sequence targeting it to caveolae/lipid rafts, but neither its targeting nor internalization depends on CAV-1 or FLOT-2, though knockdown of these proteins reduces TRAF2 expression and selectively inhibits SAPK activation.
TNFR1 localization to low-density membrane domains in human endothelial cells is mediated by a specific membrane-proximal sequence but is not required for TNF-induced NF-kappaB or SAPK activation.
TNFR1 (tumor necrosis factor receptor 1) localizes to caveolae of human endothelial-derived EA.hy926 cells. Transduced TNFR1 molecules lacking amino acid residues 229-244 (spanning the transmembrane/intercellular boundary) are expressed on the cell surface equivalently to full-length TNFR1 molecules but incompletely localize to caveolae. A peptide containing this sequence pulls down CAV-1 (caveolin-1) and TNFR1 from cell lysates but fails to do so following disruption of caveolae with methyl-beta-cyclodextrin. We previously reported that methyl-beta-cyclodextrin eliminates caveolae and blocks tumor necrosis factor (TNF)-induced internalization of TNFR1 but not TNF-induced activation of NF-kappaB in EA.hy926 cells. Both CAV-1 and FLOT-2 (flotillin-2), organizing proteins of caveolae and lipid rafts, respectively, associate with caveolae in EA.hy926 cells. Small interfering RNA-mediated knockdown of CAV-1 but not FLOT-2 strikingly reduces caveolae number. Both knockdowns reduce total TNFR1 protein expression, but neither prevents TNFR1 localization to low density membrane domains, TNF-induced internalization of TNFR1, or NF-kappaB activation by TNF. Both CAV-1 and FLOT-2 knockdowns reduce TNF-mediated activation of stress-activated protein kinase (SAPK). However, both knockdowns reduce expression of TRAF2 (TNF receptor-associated factor-2) protein, and small interfering RNA targeting of TRAF2 also selectively inhibits SAPK activation. We conclude that TNFR1 contains a membrane-proximal sequence that targets the receptor to caveolae/lipid rafts. Neither TNFR1 targeting to nor internalization from these low density membrane domains depends upon CAV-1 or FLOT-2. Furthermore, both NF-kappaB and SAPK activation appear independent of both TNFR1 localization to low density membrane domains and to TNF-induced receptor internalization.
D’Alessio et al. (2010) studied this question. siRNA knockdown of CAV-1 or FLOT-2 and TNFR1 mutations vs. Control siRNA or wild-type TNFR1 was evaluated on TNFR1 localization, internalization, and signaling (NF-κB and SAPK activation). TNFR1 contains a membrane-proximal sequence targeting it to caveolae/lipid rafts, but neither its targeting nor internalization depends on CAV-1 or FLOT-2, though knockdown of these proteins reduces TRAF2 expression and selectively inhibits SAPK activation.