Cholesterol-dependent clustering of IL-2R alpha and its colocalization with HLA and CD48 on T lymphoma cells suggest their functional association with lipid rafts

Immunogold staining and electron microscopy show that IL-2 receptor alpha-s ubunits exhibit nonrandom surface distribution on human T lymphoma cells, A nalysis of interparticle distances reveals that this clustering on the scal e of a few hundred nanometers is independent of the presence of IL-2 and of the expression of the IL-2R beta-subunit. Clustering of IL-2R alpha is con firmed by confocal microscopy, yielding the same average cluster size, appr oximate to 600-800 nm, as electron microscopy. HLA class I and II and CD48 molecules also form clusters of the same size. Disruption of cholesterol-ri ch lipid rafts with filipin or depletion of membrane cholesterol with methy l-beta-cyclodextrin results in the blurring of cluster boundaries and an ap parent dispersion of clusters for all four proteins. Interestingly, the tra nsferrin receptor which is thought to be located outside lipid rafts, exhib its clusters that are only 300 nm in size and are less affected by modifyin g the membrane cholesterol content. Furthermore, transferrin receptor clust ers hardly colocalize with IL-2R alpha, HLA, and CD48 molecules (crosscorre lation coefficient is 0.05), whereas IL-2R alpha colocalizes with both HLA and CD48 (crosscorrelation coefficient is between 0.37 and 0.46), This cocl ustering is confirmed by electron microscopy. The submicron clusters of IL- 2R alpha chains and their coclustering with HLA and CD48, presumably associ ated with lipid rafts, could underlie the efficiency of signaling in lympho id cells.