Targeting of tumor necrosis factor receptor 1 to low density plasma membrane domains in human endothelial cells.
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.
Interdepartmental Program in Vascular Biology and Therapeutics, Yale University School of Medicine, New Haven, CT 06520-8089, USA.
Alessio
A
D'Alessio
Martin S
MS
Kluger
Jie H
JH
Li
Rafia
R
Al-Lamki
John R
JR
Bradley
Jordan S
JS
Pober
D021941:Caveolae / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002469:Cell Separation; D042783:Endothelial Cells / Q000166:cytology*; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry; D048055:Mitogen-Activated Protein Kinase 8 / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D034741:RNA, Small Interfering / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism*; D015398:Signal Transduction; D047392:beta-Cyclodextrins / Q000737:chemistry
D016328:NF-kappa B; D010455:Peptides; D034741:RNA, Small Interfering; D047888:Receptors, Tumor Necrosis Factor, Type I; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D048055:Mitogen-Activated Protein Kinase 8