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...

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Bibliographic Details
Published in:The Journal of biological chemistry 2010-07, Vol.285 (31), p.23868-23879
Main Authors: D'Alessio, Alessio, Kluger, Martin S., Li, Jie H., Al-Lamki, Rafia, Bradley, John R., Pober, Jordan S.
Format: Article
Language:English
Subjects:
beta-Cyclodextrins - chemistry
Caveolae
Caveolae - metabolism
Cell Biology
Cell Membrane - metabolism
Cell Separation
Endothelial Cells - cytology
Flow Cytometry
Fluorescent Antibody Technique, Indirect
Humans
Lipid Raft
MAP Kinases (MAPKs)
Membrane Biology
Membrane Microdomains - chemistry
Mitogen-Activated Protein Kinase 8 - metabolism
NF-kappa B
NF-kappa B - metabolism
Peptides - chemistry
Receptors, Tumor Necrosis Factor, Type I - metabolism
RNA, Small Interfering - metabolism
Signal Transduction
Tumor Necrosis Factor (TNF)
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Summary: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-β-cyclodextrin. We previously reported that methyl-β-cyclodextrin eliminates caveolae and blocks tumor necrosis factor (TNF)-induced internalization of TNFR1 but not TNF-induced activation of NF-κB 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-κB 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-κB and SAPK activation appear independent of both TNFR1 localization to low density membrane domains and to TNF-induced receptor internalization.
ISSN:0021-9258
1083-351X
1083-351X
DOI:10.1074/jbc.M110.122853