Separation of a cholesterol-enriched microdomain involved in T-cell signal transduction.
We isolated a cholesterol-enriched membrane subpopulation from the so-called lipid raft fractions of Jurkat T-cells by taking advantage of its selective binding to a cholesterol-binding probe, BCtheta. The BCtheta-bound membrane subpopulation has a much higher cholesterol/phospholipid (C/P) molar ratio (approximately 1.0) than the BCtheta-unbound population in raft fractions (approximately 0.3). It contains not only the raft markers GM1 and flotillin, but also some T-cell receptor (TCR) signalling molecules, including Lck, Fyn and LAT. In addition, Csk and PAG, inhibitory molecules of the TCR signalling cascade, are also contained in the BCtheta-bound membranes. On the other hand, CD3epsilon, CD3zeta and Zap70 are localized in the BCtheta-unbound membranes, segregated from other TCR signalling molecules under nonstimulated conditions. However, upon stimulation of TCR, portions of CD3epsilon, CD3zeta and Zap70 are recruited to the BCtheta-bound membranes. The Triton X-100 concentration used for lipid raft preparation affects neither the C/P ratio nor protein composition of the BCtheta-bound membranes. These results show that our method is useful for isolating a particular cholesterol-rich membrane domain of T-cells, which could be a core domain controlling the TCR signalling cascade.
Shimada
Yukiko
Y
Biomembrane Research Group, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo, Japan. yshimada@tmig.or.jp
Inomata
Mitsushi
M
Suzuki
Hidenori
H
Hayashi
Masami
M
Waheed
A Abdul
AA
Ohno-Iwashita
Yoshiko
Y
D000906:Antibodies / Q000276:immunology; D015415:Biomarkers; D017252:CD3 Complex / Q000276:immunology / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D017830:Octoxynol / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects; D013601:T-Lymphocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000378:metabolism
D000906:Antibodies; D015415:Biomarkers; D017252:CD3 Complex; D017830:Octoxynol; D002784:Cholesterol; D051746:ZAP-70 Protein-Tyrosine Kinase