Many surface receptors and signaling molecules are thought to associate with unique membrane microdomains termed lipid rafts. We examined the Involvement of lipid rafts in the activation of leukocyte function-associated antigen-1 (LFA-1). Depletion or sequestration of cholesterol with methyl-beta-cyclodextrin (MCD) or filipin, respectively, strongly inhibited LFA-1-mediated adhesion of T-cell lines and primary T cells. This inhibition was reversed by cholesterol reconstitution. LIFA-1 on T-cell lines was detected in cold Triton X-100-insoluble lipid rafts, which were disrupted by MCD or fillpin treatment. However, no LIFA-1 on primary T cells was detected in lipid rafts isolated by the same procedures, and these rafts were resistant to cholesterol depletion or sequestration. Association of LIFA-1 with lipid rafts of primary T cells could be detected only when they were isolated with another nonionic detergent, Brij 35. Upon treatment with MCD, LIFA-1 in Brij 35-insoluble lipid rafts partially shifted to nonraft fractions. T-cell lines were found to have a high level of cholesterol and a low level of ganglioside GM1, a common marker for lipid rafts, whereas primary T cells have a much lower level of cholesterol and a very high amount of GM1. Cross-linking of LFA-1 on primary T cells induced cocapping of cholesterol but not GM1. These results suggest that lipid rafts of T cells are heterogenous, and LFA-1 associates with a subset of lipid rafts containing a high level of cholesterol. This association seems to regulate LFA-1 functions, possibly by facilitating LFA-1 clustering. (C) 2003 by The American Society of Hematology.
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