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Separation of a cholesterol‐enriched microdomain involved in T‐cell signal transduction
Author(s) -
Shimada Yukiko,
Inomata Mitsushi,
Suzuki Hidenori,
Hayashi Masami,
Abdul Waheed A.,
OhnoIwashita Yoshiko
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.04938.x
Subject(s) - lipid raft , t cell receptor , lipid microdomain , jurkat cells , microbiology and biotechnology , biology , fyn , zap70 , t cell , signal transduction , raft , membrane , biochemistry , chemistry , proto oncogene tyrosine protein kinase src , immunology , immune system , polymer , organic chemistry , copolymer
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, BCθ. The BCθ‐bound membrane subpopulation has a much higher cholesterol/phospholipid (C/P) molar ratio (≈ 1.0) than the BCθ‐unbound population in raft fractions (≈ 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 BCθ‐bound membranes. On the other hand, CD3ε, CD3ζ and Zap70 are localized in the BCθ‐unbound membranes, segregated from other TCR signalling molecules under nonstimulated conditions. However, upon stimulation of TCR, portions of CD3ε, CD3ζ and Zap70 are recruited to the BCθ‐bound membranes. The Triton X‐100 concentration used for lipid raft preparation affects neither the C/P ratio nor protein composition of the BCθ‐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.