Targeting of tumor necrosis factor receptor 1 to low density plasma membrane domains in human endothelial cells

Alessio D'Alessio, Ms Kluger, Jh Li, R Al Lamki, Jr Bradley, Js Pober

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

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.
Original languageEnglish
Pages (from-to)23868-23879
Number of pages12
JournalJOURNAL OF BIOLOGICAL CHEMISTRY
Volume285
DOIs
Publication statusPublished - 2010

Keywords

  • Caveolae
  • Cell Membrane
  • Cell Separation
  • Endothelial Cells
  • Fluorescent Antibody Technique, Indirect
  • Membrane Microdomains
  • Mitogen-Activated Protein Kinase 8
  • NF-kappa B
  • Peptides
  • RNA, Small Interfering
  • Receptors, Tumor Necrosis Factor, Type I
  • Signal Transduction
  • beta-Cyclodextrins

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