Subunits (a, b and c) of the interleukin-2 receptor complex (IL-2R) are involved in both proliferative and activationinduced cell death (AICD) signaling of T cells. In addition, the signaling b and c chains are shared by other cytokines (e.g. . However, the molecular mechanisms responsible for recruiting/sorting the a chains to the signaling chains at the cell surface are not clear. Here we show, in four cell lines of human adult T cell lymphoma/leukemia origin, that the three IL-2R subunits are compartmented together with HLA glycoproteins and CD48 molecules in the plasma membrane, by means of fluorescence resonance energy transfer (FRET), confocal microscopy and immunobiochemical techniques. In addition to the b and c c chains constitutively expressed in detergent-resistant membrane fractions (DRMs) of T cells, IL-2Ra (CD25) was also found in DRMs, independently of its ligand-occupation. Association of CD25 with rafts was also confirmed by its colocalization with GM-1 ganglioside. Depletion of membrane cholesterol using methyl-b-cyclodextrin substantially reduced co-clustering of CD25 with CD48 and HLA-DR, as well as the IL-2 stimulated tyrosine-phosphorylation of STATs (signal transducer and activator of transcription). These data indicate a GPI-microdomain (raft)-assisted recruitment of CD25 to the vicinity of the signaling b and c c chains. Rafts may promote rapid formation of a high affinity IL-2R complex, even at low levels of IL-2 stimulus, and may also form a platform for the regulation of IL-2 induced signals by GPI-proteins (e.g. CD48). Based on these data, the integrity of these GPI-microdomains seems critical in signal transduction through the IL-2R complex. Recent FRET data, in contrast to an earlier Ôsequential subunit-organizationÕ (affinity conversion) model [10], suggested a preassembly of the three IL-2R subunits, even in the absence of their relevant cytokine ligands in the plasma membrane of T lymphoma cells. Binding of the physiological ligands (IL-2, IL-7, IL-15) was reported to selectively modulate the mutual molecular proximities/ interactions of the IL-2R a, b and c c chains [11]. Microscopic (confocal fluorescence and immunogold labeling-based electron microscopy) studies revealed large scale (% 4-800 nm) overlapping clusters of CD25 and HLA molecules on T cell lines [12]. These observations all suggest that the above membrane proteins are somewhat compartmentalized in T cell plasma membranes.

Distribution and lateral organization of Kv1.3 potassium channels and CD3 molecules were studied by using electron microscopy, confocal laser scanning microscopy, and fluorescence resonance energy transfer. Immunogold labeling and electron microscopy showed that the distribution of FLAG epitope-tagged Kv1.3 channels (Kv1.3͞FLAG) significantly differs from the stochastic Poisson distribution in the plasma membrane of human T lymphoma cells. Confocal laser scanning microscopy images showed that Kv1.3͞FLAG channels and CD3 molecules accumulated in largely overlapping membrane areas. The numerical analysis of crosscorrelation of the spatial intensity distributions yielded a high correlation coefficient (C ‫؍‬ 0.64). A different hierarchical level of molecular proximity between Kv1.3͞FLAG and CD3 proteins was reported by a high fluorescence resonance energy transfer efficiency (E ‫؍‬ 51%). These findings implicate that reciprocal regulation of ion-channel activity, membrane potential, and the function of receptor complexes may contribute to the proper functioning of the immunological synapse.