Lipid Rafts as Platforms for Cytokine Receptor Assembly and Signaling

Interleukin-2 (IL-2) and -15 (IL-15) are substantially involved in controlling T cell homeostasis and function [119,120,121]. Their receptors comprise three distinct components: while the a-chains are cytokine-specific, the b- and gc-subunits are employed by both IL-2 and IL-15. In addition, the so-called common gc-chain is the component of a series of other cytokine receptors - that is, the members of the gc cytokine receptor family (IL-4, IL-7, IL-9, and IL-21). As a result of combining various subunits, several forms of receptor complexes with different affinities may exist at the cell surface. Heterodimerization of the intracellular domains of the band gc-chains was found critical for one set of signaling events shared by both cytokines. In this case the two cytokines activate similar signaling pathways involving Janus kinase (JAK1 and JAK3)-assisted tyrosine phosphorylation of downstream signaling molecules (e.g., STAT3 and STAT5). Sharing of common receptor subunits explains the redundancy in the biological actions of IL-2 and IL-15 (e.g., stimulation of T cell proliferation) [119,120]. In addition to the shared functions, they can also exhibit opposing contributions to T-cell-mediated immunity [119,120]. Since IL-2 plays a pivotal role in activation-induced cell death (AICD), it is critical in the elimination of self-reactive T cells and so in peripheral self-tolerance. At the same time, IL-15 manifests anti-apoptotic actions and inhibits IL-2-mediated AICD. IL-2 and IL-15 play opposing roles in the control of the homeostasis of CD8+ memory phenotype T cells. IL-15 provides potent and selective stimulation of memory phenotype CD8+ T cells in vivo, whereas IL-2 inhibits the persistence of these cells.

FRET data suggested that, in contrast to the earlier "sequential subunit-organi-zation" (affinity conversion) model proposing ligand-induced association of initially separate subunits [122], the three chains of the high-affinity IL-2R (IL-2Rabgc) complex are preassembled in the plasma membrane of human T lymphoma cells even in the absence of IL-2 [123]. Binding of IL-2 or IL-15 was reported to differentially modulate the conformation of the receptor complex: addition of IL-2 made the heterotrimer more compact, while IL-15 loosened the interaction/ proximity between the IL-2Ra and gc-chains [123]. A similar preassembly of the heterotrimeric IL-15R (IL-15Rabgc), as well as the molecular proximity of IL-15Ra and IL-2Ra was demonstrated on human T cells expressing all the elements of the IL-2/IL-15 receptor system [98] (Fig. 7.3). Whereas neither IL-2, nor IL-15 affected the molecular association of the two a-chains, the interaction between the b- and IL-15Ra subunits became tighter upon IL-15 treatment, as indicated by FRET measurements. Based on these data, a heterotetrameric model of the IL-2/IL-15R complex can be envisaged: binding of IL-2 or IL-15 rearranges the subunits to form the appropriate abgc high-affinity receptor complex, while the other a-chain rotates or moves away from the site of cytokine-receptor interaction. FRET experiments also indicated the homodimeric/oligomeric molecular association of IL-15Ra [98]. Homoassociation of IL-2Ra may also occur in T lymphoma cells, although in a cell type-dependent manner [124].

It was shown by using biophysical (FRET and CLSM) and biochemical approaches that IL-2/IL-15R subunits are mainly partitioned into lipid rafts in the plasma membrane of human T lymphoma cells [39,79,98] (see Fig. 7.3). These rafts contained, among others, GM1 gangliosides, the GPI-anchored CD48 protein and a major fraction of MHC glycoproteins. At the same time the domains containing transferrin receptors (coated pits) were clearly distinct from GM1-contain-ing lipid rafts (Fig. 7.4).

In addition, the high-affinity IL-2R was shown to be resistant to cold detergent extraction both in murine and human T lymphoma cells [79,125]. It was also demonstrated that the integrity of lipid rafts has a crucial role in organizing the lateral distribution of IL-2R and it is also essential for IL-2-mediated signaling [39,79]. Disruption of the native structure of lipid rafts by cholesterol extraction resulted in m m vt

FRET efficiency (%)

the dispersion of the above raft components and simultaneously abrogated STAT3/ STAT5 tyrosine phosphorylation related to IL-2 signaling [39,79]. This suggests that raft integrity is critical in keeping the intracellular domains of the b- and gc-chains together with the docked JAKs and STATs in a proper, juxtaposed position. Upon cholesterol depletion, the size of lipid rafts increased and the boundaries of the microdomains became fuzzier - that is, the compactness and cohesion within the microdomain declined [39]. This implies that the lipid microenvironment, in particular cholesterol, might be an important factor in maintaining the integrity of signaling complexes. Lipid rafts may promote the formation and cytokine-specific modulation of IL-2R/IL-15R complexes and b- and gc-subunit "switching" between IL-2 and IL-15 receptors as well.

Marmor and Julius proposed a different mode of IL-2R signal regulation by rafts, based on observations on murine T cells [126]. Whereas IL-2Ra was also found constitutively enriched in lipid rafts, the b- and gc-chains, along with JAK1 and JAK3 kinases, were found mostly in the detergent-soluble membrane fractions. IL-2-mediated assembly of the high-affinity receptor complex as well as activation of JAKs occurred exclusively in the soluble fractions. As a consequence, disruption of lipid raft integrity did not impair IL-2R-induced signaling. It was proposed that sequestration of IL-2Ra within raft domains of murine T cells hampers its interactions and regulates IL-2 signaling through impeding its interaction with the signaling bgc heterodimer.

A third mechanism for raft-assisted IL-2 signaling is outlined by Goebel et al. [127]. Using biochemical approaches, these authors demonstrated selective enrichment of IL-2/15Rb chains, but not cytokine-specific a-chains or gc-chains, in lipid rafts of phytohemagglutinin (PHA)-activated human peripheral T cells. IL-2 stimulation was accompanied by a partial translocation of the b-chains along with the associated signaling molecules (JAK1, lck, Grb-2) to the soluble membrane fraction. Furthermore, disruption of lipid raft integrity attenuated IL-2 signaling.

O Fig. 7.3 Co-localization of IL-15Ra and IL-2Ra in lipid rafts of FT7.10 T lymphoma cells. (Panels A-D): Confocal microscopic images of the distribution of IL-15Ra (panel A, Cy3-anti-FLAG), IL-2Ra (panel B, Cy5-7G7 B6) and GM1 ganglioside, a lipid raft marker (panel C, labeled by Alexa Fluor 488-cholera toxin B). In the overlay image (panel D) those pixels appearing in white represent co-localization of all three molecules. Confocal sections were recorded from the cover slip-proximal surface of the cell. Pairwise correlation coefficients between the different channels were fairly high (Cab = 0.79, Cac = 0.59, Cbc = 0.67), referring to substantial overlap between the lateral distributions of the studied molecules. (Scale bar= 2 mm.) Panels E-H: Acceptor photobleaching FRET measure ment: Confocal images of IL-15Ra (panel E) and IL-2Ra (panel F) recorded before acceptor photobleaching (see Section 7.2 for a description of FRET microscopic methods). Receptor subunits were targeted by Cy3-7A4 24 (donor) and Cy5-anti-Tac (acceptor) mAbs. Panel G shows the pixel-by-pixel FRET efficiency map between IL-15Ra and IL-2Ra as determined from the donor images taken before and after photobleaching. The color code ranges between FRET efficiency values of 0 (purple) to 100% (red). Pixels with fluorescence intensities lower than the background threshold are displayed in black. Panel H shows the frequency distribution histogram of FRET efficiencies in the individual pixels. The mean FRET efficiency value was 17%.

Fig. 7.4 Confocal laser scanning microscopy (CLSM) images of the distribution of transferrin receptors and GM1 gangliosides. Transferrin receptors (B), which are enriched in coated pits, were co-localized poorly with GM1 gangliosides segregated into lipid rafts (A). The distinctness of the distributions (C) clearly indicated segregation of the two types of membrane domains. The correlation coefficient between the images was C = 0.22. Transferrin receptors were labeled by Cy3-MEM75 mAbs, and GM1 was targeted by Cy5-cholera toxin B.

Fig. 7.4 Confocal laser scanning microscopy (CLSM) images of the distribution of transferrin receptors and GM1 gangliosides. Transferrin receptors (B), which are enriched in coated pits, were co-localized poorly with GM1 gangliosides segregated into lipid rafts (A). The distinctness of the distributions (C) clearly indicated segregation of the two types of membrane domains. The correlation coefficient between the images was C = 0.22. Transferrin receptors were labeled by Cy3-MEM75 mAbs, and GM1 was targeted by Cy5-cholera toxin B.

These authors suggested that sequestration of the b-chains in rafts prior to IL-2 stimulation controls the specific cytokine responsiveness, since it ensures that pairing with the more "promiscuous" gc-chains only takes place upon binding of the appropriate ligand [127].

Association of IL-2 receptors with detergent-resistant membrane microdomains was also reported to define a clathrin-independent endocytotic pathway [128].

Incongruence observed in the raft association of the IL-2/IL-15R system could indicate that composition of rafts as well as partition of a given protein between the raft and non-raft membrane regions can exhibit cell- or species-specificity [79,127]. Whether the observed discrepancies are only due to the different experimental approaches or they are caused by real differences in the observed systems (and if yes, what is the cause of distinct localization: post-translational modification, lipid composition or protein-protein interactions) is yet to be determined.

Flow cytometric FRET experiments revealed the co-localization of the IL-2/IL-15R system and MHC I molecules in the plasma membrane of cells of human T lymphoma/leukemia origin [39,79,98]. Although the exact role of this co-localiza-

Fig. 7.5 Mobility and co-mobility measurements by FCS and FCCS. (A) Normalized autocorrelation curves detected from Cy5-tagged anti-Tac Fabs free in solution or bound to IL-2Ra subunits on Kit 225 K6 T lymphoma cells. The diffusion time of the antibody decreased by an order of magnitude upon receptor binding. (B) Cross-correlation curve measured on Kit 225 FT 7.10 cells between IL-2Ra and IL-15Ra (labeled with Cy5-anti-Tac Fab and Alexa 488 anti-FLAG mAbs,

Fig. 7.5 Mobility and co-mobility measurements by FCS and FCCS. (A) Normalized autocorrelation curves detected from Cy5-tagged anti-Tac Fabs free in solution or bound to IL-2Ra subunits on Kit 225 K6 T lymphoma cells. The diffusion time of the antibody decreased by an order of magnitude upon receptor binding. (B) Cross-correlation curve measured on Kit 225 FT 7.10 cells between IL-2Ra and IL-15Ra (labeled with Cy5-anti-Tac Fab and Alexa 488 anti-FLAG mAbs, respectively). The nonzero cross-correlation amplitude suggests that at least a certain fraction of the proteins form stable complexes for at least the duration of the diffusion time. As a negative control, cross-correlation between transferrin receptors (Trfr) and IL-2Ra was determined on K6 cells yielding a flat correlation curve, which is indicative of no interaction. Receptors were labeled by Alexa 488-MEM75 mAb and Cy5-anti-Tac Fab, respectively.

tion has not yet been elucidated, a regulatory tyrosine phosphorylation cross-talk, as suggested earlier for the class I MHC-insulin receptor interaction [15], cannot be excluded [39,79]. Association of the IL-2/IL-15R with MHC II glycoproteins was also demonstrated. Co-localization of the elements of the IL-2/IL-15R system with MHC glycoproteins also takes place in lipid rafts, as revealed by confocal microscopy [39,98].

FRET assays do not report on the dynamics and stability of protein-protein interactions. By using FCCS, it was shown that a subunits of IL-2R and IL-15R diffused together at least for several tens of milliseconds - the time window of an FCCS experiment (Fig. 7.5). Similar stable association was detected between MHC I and IL-2Ra or IL-15Ra chains [98]. On the other hand, no cross-correlation could be detected between IL-2Ra and coated pit-located transferrin receptor molecules (Fig. 7.5).

An interesting consequence of these results follows from the relative expression levels of the studied molecules. The ratio of the amount of IL-15Ra, IL-2Ra and MHC I is -1:10:50-100 on the Kit 225 FT7.10 T lymphoma cells used in the FCCS experiments. If complexes of 1:1 stoichiometry were formed between IL-15Ra and MHC I, then the out-of-complex fraction of MHC I molecules would suppress the cross-correlation amplitude below the detection level (see Eq. 7.8). This suggests that higher-order aggregates of MHC class I molecules float together with IL-2 and IL-15 receptors in large supramolecular complexes in the plasma membrane. These results are in accordance with previous data on the homoassociation of MHC I molecules detected by FRET and electron microscopy/atomic force microscopy (see Section 7.3) [59,83].

Our observations suggest the possibility of a supramolecular complex of MHC, ICAM-1 molecules and cytokine receptor subunits that could include all members of the gc cytokine receptor family in addition to IL-2Ra and IL-15Ra, in particular, IL-4Ra, IL-7Ra, IL-9Ra and IL-21Ra. Such an association in a lipid raft-accommodated supercomplex could provide one explanation for the functional competition among cytokines that has been observed on the simultaneous addition of IL-2 and IL-4 to lymphocytes. Furthermore, the definition of such a supercomplex of cytokine receptors would also add to our understanding of the regulation of lymphocyte proliferation and effector immune responses that are mediated by these pivotal gc-associated cytokines.

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