TY - JOUR
T1 - Targeting of tumor necrosis factor receptor 1 to low density plasma membrane domains in human endothelial cells
AU - D'Alessio, Alessio
AU - 33186,
AU - FACOLTA', DI MEDICINA E CHIRURGIA "A.GEMELLI"
AU - pubblica, ROMA - Dipartimento di Scienze della vita e sanità
AU - Kluger,
AU - Li, Jh
AU - Al Lamki, R
AU - Bradley, Jr
AU - Pober, Js
PY - 2010
Y1 - 2010
N2 - 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.
AB - 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.
KW - Caveolae
KW - Cell Membrane
KW - Cell Separation
KW - Endothelial Cells
KW - Fluorescent Antibody Technique, Indirect
KW - Membrane Microdomains
KW - Mitogen-Activated Protein Kinase 8
KW - NF-kappa B
KW - Peptides
KW - RNA, Small Interfering
KW - Receptors, Tumor Necrosis Factor, Type I
KW - Signal Transduction
KW - beta-Cyclodextrins
KW - Caveolae
KW - Cell Membrane
KW - Cell Separation
KW - Endothelial Cells
KW - Fluorescent Antibody Technique, Indirect
KW - Membrane Microdomains
KW - Mitogen-Activated Protein Kinase 8
KW - NF-kappa B
KW - Peptides
KW - RNA, Small Interfering
KW - Receptors, Tumor Necrosis Factor, Type I
KW - Signal Transduction
KW - beta-Cyclodextrins
UR - http://hdl.handle.net/10807/16800
U2 - 10.1074/jbc.M110.122853
DO - 10.1074/jbc.M110.122853
M3 - Article
VL - 285
SP - 23868
EP - 23879
JO - JOURNAL OF BIOLOGICAL CHEMISTRY
JF - JOURNAL OF BIOLOGICAL CHEMISTRY
SN - 1083-351X
ER -