The alpha1a-adrenergic receptor occupies membrane rafts with its G protein effectors but internalizes via clathrin-coated pits

The alpha(1a)-adrenergic receptor (alpha(1a)AR) occupies intracellular and plasma membranes in both native and heterologous expression systems. Based on multiple independent lines of evidence, we demonstrate the alpha(1a)AR at the cell surface occupies membrane rafts but exits from rafts following stimulation. In non-detergent raft preparations, basal alpha(1a)AR is present in low density membrane rafts and colocalizes with its G protein effectors on density gradients. Raft disruption by cholesterol depletion with methyl-beta-cyclodextrin eliminates these light rafts. To confirm the presence of the alpha(1a)AR in plasma membrane rafts, fluorescence resonance energy transfer measurements were used to demonstrate colocalization of surface receptor and the raft marker, cholera toxin B. This colocalization was largely lost following alpha(1a)AR stimulation with phenylephrine. Similarly, receptor stimulation causes exit of the alpha(1a)AR from light rafts within 3-10 min in contrast to the G proteins, which largely remain in light rafts. Importantly, this delayed exit of the alpha(1a)AR suggests acute receptor signaling and desensitization occur entirely within rafts. Interestingly, both confocal analysis and measurement of surface alpha(1a)AR levels indicate modest receptor internalization during the 10 min following stimulation, suggesting most of the receptor has entered non-raft plasma membrane. Nevertheless, activation does increase the rate of receptor internalization as does disruption of rafts with methyl-beta-cyclodextrin, suggesting raft exit enables internalization. Confocal analysis of surface-labeled hemagglutinin-alpha(1a)AR reveals that basal and stimulated receptor occupies clathrin pits in fixed cells consistent with previous indirect evidence. The evidence presented here strongly suggests the alpha(1a)AR is a lipid raft protein under basal conditions and implies agonist-mediated signaling occurs from rafts.     

Integrin leukocyte function-associated antigen-1-mediated cell binding can be activated by clustering of membrane rafts

The leukocyte function-associated antigen-1 (LFA-1) integrin (CD11a/CD18) is an important adhesion molecule for lymphocyte migration and the initiation of an immune response. At the cell surface, LFA-1 activity can be regulated by divalent cations that enhance receptor affinity but also by membrane clustering induced by treatment of cells with substances such as phorbol esters. Membrane clustering leads to increased LFA-1 avidity. We report here that LFA-1-mediated binding of mouse thymocytes or activated T lymphocytes to intercellular adhesion molecule 1 can be rapidly induced by clustering of membrane rafts using antibodies to the glycosylphophatidylinositol-anchored molecule CD24 or cholera toxin (CTx). CD24 and CD18 were found to co-localize in rafts and cross-linking with CTx lead to enhanced LFA-1 clustering. We observed that disruption of raft integrity by lowering the membrane cholesterol content abolished the CTx and the phorbol 12-myristate 13-acetate-induced LFA-1 binding but left the ability to activate LFA-1 with Mg(2+)/EGTA unimpaired. In contrast to activation with Mg(2+)/EGTA, activation via raft clustering was dependent on PI3-kinase, required cytoskeletal mobility, and was accompanied by Tyr phosphorylation of a 18-kDa protein. Our results support the notion that rafts as preformed adhesion platforms could be important for the rapid regulation of lymphocyte adhesion.     

Cold Induces Micro- and Nano-Scale Reorganization of Lipid Raft Markers at Mounds of T-Cell Membrane Fluctuations

Whether and how cold causes changes in cell-membrane or lipid rafts remain poorly characterized. Using the NSOM/QD and confocal imaging systems, we found that cold caused microscale redistribution of lipid raft markers, GM1 for lipid and CD59 for protein, from the peripheral part of microdomains to the central part on Jurkat T cells, and that cold also induced the nanoscale size-enlargement (1/3- to 2/3-fold) of the nanoclusters of lipid raft markers and even the colocalization of GM1 and CD59 nanoclusters. These findings indicate cold-induced lateral rearrangement/coalescence of raft-related membrane heterogeneity. The cold-induced re-distribution of lipid raft markers under a nearly-natural condition provide clues for their alternations, and help to propose a model in which raft lipids associate themselves or interact with protein components to generate functional membrane heterogeneity in response to stimulus. The data also underscore the possible cold-induced artifacts in early-described cold-related experiments and the detergent-resistance-based analyses of lipid rafts at 4°C, and provide a biophysical explanation for recently-reported cold-induced activation of signaling pathways in T cells. Importantly, our fluorescence-topographic NSOM imaging demonstrated that GM1/CD59 raft markers distributed and re-distributed at mounds but not depressions of T-cell membrane fluctuations. Such mound-top distribution of lipid raft markers or lipid rafts provides spatial advantage for lipid rafts or contact molecules interacting readily with neighboring cells or free molecules.     

Triglyceride-rich lipoprotein lipolysis increases aggregation of endothelial cell membrane microdomains and produces reactive oxygen species

Triglyceride-rich lipoprotein (TGRL) lipolysis may provide a proinflammatory stimulus to endothelium. Detergent-resistant plasma membrane microdomains (lipid rafts) have a number of functions in endothelial cell inflammation. The mechanisms of TGRL lipolysis-induced endothelial cell injury were investigated by examining endothelial cell lipid rafts and production of reactive oxygen species (ROS). Lipid raft microdomains in human aortic endothelial cells were visualized by confocal microscopy with fluorescein isothiocyanate-labeled cholera toxin B as a lipid raft marker. Incubation of Atto565-labeled TGRL with lipid raft-labeled endothelial cells showed that TGRL colocalized with the lipid rafts, TGRL lipolysis caused clustering and aggregation of lipid rafts, and colocalization of TGRL remnant particles on the endothelial cells aggregated lipid rafts. Furthermore, TGRL lipolysis caused translocation of low-density lipoprotein receptor-related protein, endothelial nitric oxide synthase, and caveolin-1 from raft regions to nonraft regions of the membrane 3 h after treatment with TGRL lipolysis. TGRL lipolysis significantly increased the production of ROS in endothelial cells, and both NADPH oxidase and cytochrome P-450 inhibitors reduced production of ROS. Our studies suggest that alteration of lipid raft morphology and composition and ROS production could contribute to TGRL lipolysis-mediated endothelial cell injury.     

Lipid rafts: cell surface platforms for T cell signaling

The Src family tyrosine kinase Lck is essential for T cell development and T cell receptor (TCR) signaling. Lck is post-translationally fatty acylated at its N-terminus conferring membrane targeting and concentration in plasma membrane lipid rafts, which are lipid-based organisational platforms. Confocal fluorescence microscopy shows that Lck colocalizes in rafts with GPI-linked proteins, the adaptor protein LAT and Ras, but not with non-raft membrane proteins including the protein tyrosine phosphatase CD45. The TCR also associates with lipid rafts and its cross-linking causes coaggregation of raft-associated proteins including Lck, but not of CD45. Cross-linking of either the TCR or rafts strongly induces specific tyrosine phosphorylation of the TCR in the rafts. Remarkably, raft patching alone induces signalling events analogous to TCR stimulation, with the same dependence on expression of key TCR signalling molecules. Our results indicate a mechanism whereby TCR engagement promotes aggregation of lipid rafts, which facilitates colocalization of signaling proteins including Lck, LAT, and the TCR, while excluding CD45, thereby potentiating protein tyrosine phosphorylation and downstream signaling. We are currently testing this hypothesis as well as using imaging techniques such as fluorescence resonance energy transfer (FRET) microscopy to study the dynamics of proteins and lipids in lipid rafts in living cells undergoing signaling events. Recent data show that the key phosphoinositide PI(4,5)P2 is concentrated in T cell lipid rafts and that on stimulation of the cells it is rapidly converted to PI(3,4,5)P3 and diacylglycerol within rafts. Thus rafts are hotspots for both protein and lipid signalling pathways.     

Lipid rafts prepared by different methods contain different connexin channels, but gap junctions are not lipid rafts

Cell extraction with cold nonionic detergents or alkaline carbonate prepares an insoluble membrane fraction whose buoyant density permits its flotation in discontinuous sucrose gradients. These lipid "rafts" are implicated in protein sorting and are attractive candidates as platforms that coordinate signal transduction pathways with intracellular substrates. Gap junctions form a direct molecular signaling pathway by end-to-end apposition of hemichannels containing one (homomeric) or more (heteromeric) connexin isoforms. Residency of channels composed of Cx26 and/or Cx32 in lipid rafts was assessed by membrane insolubility in alkaline carbonate or different concentrations of Triton X100, Nonidet P40 and Brij-58 nonionic detergents. Using Triton X100, insoluble raft membranes contained homomeric Cx32 channels, but Cx26-containing channels only when low detergent concentrations were used. Results were similar using Nonidet P40, except that Cx26-containing channels were excluded from raft membranes at all detergent concentrations. In contrast, homomeric Cx26 channels were enriched within Brij-58-insoluble rafts, whereas Cx32-containing channels partitioned between raft and nonraft membranes. Immunofluorescence microscopy showed prominent colocalization only of nonjunctional connexin channels with raft plasma membrane; junctional plaques were not lipid rafts. Rafts prepared by different extraction methods had considerable quantitative and qualitative differences in their lipid compositions. That functionally different nonjunctional connexin channels partition among rafts with distinct lipid compositions suggests that unpaired Cx26 and/or Cx32 channels exist in membrane domains of slightly different physicochemical character. Rafts may be involved in trafficking of plasma membrane connexin channels to gap junctions.     

Human immunodeficiency virus type 1 virological synapse formation in T cells requires lipid raft integrity

Human immunodeficiency virus type 1 (HIV-1) can spread directly between T cells by forming a supramolecular structure termed a virological synapse (VS). HIV-1 envelope glycoproteins (Env) are required for VS assembly, but their mode of recruitment is unclear. We investigated the distribution of GM1-rich lipid rafts in HIV-1-infected (effector) T cells and observed Env colocalization with polarized raft markers GM1 and CD59 but not with the transferrin receptor that is excluded from lipid rafts. In conjugates of effector T cells and target CD4+ T cells, GM1, Env, and Gag relocated to the cell-cell interface. The depletion of cholesterol in the infected cell dispersed Env and GM1 within the plasma membrane, eliminated Gag clustering at the site of cell-cell contact, and abolished assembly of the VS. Raft integrity is therefore critical for Env and Gag co-clustering and VS assembly in T-cell conjugates.     

CD4 ligation excludes the Carma1-Bcl10-MALT1 complex from GM1-positive membrane rafts in CD3/CD28 activated T cells

The antibody 13B8.2, which is directed against the CDR3-like loop on the D1 domain of CD4, induces CD4/ZAP-70 reorganization and ceramide release in membrane rafts. Here, we investigated whether CD4/ZAP-70 compartmentalization could be mediated by an effect of 13B8.2 on the Carma1–Bcl10–MALT1 complex in membrane rafts. We report that treatment of CD3/CD28-activated Jurkat T cells with 13B8.2, but not rituximab, excluded Carma1–Bcl10–MALT1 proteins from GM1⁺ membrane rafts and concomitantly decreased NF-κB activation. Fluorescence confocal imaging confirmed that Carma1–Bcl10 and Carma1-MALT1 co-patching, observed in GM1⁺ membrane rafts following CD3/CD28 activation, were abrogated after a 24 h-treatment with 13B8.2. The CD4/ZAP-70 compartmentalization in membrane rafts induced by 13B8.2 is thus related to Carma1–Bcl10–MALT1 raft exclusion.     

Vav1/Rac-dependent actin cytoskeleton reorganization is required for lipid raft clustering in T cells.

Formation of the immunological synapse (IS) in T cells involves large scale molecular movements that are mediated, at least in part, by reorganization of the actin cytoskeleton. Various signaling proteins accumulate at the IS and are localized in specialized membrane microdomains, known as lipid rafts. We have shown previously that lipid rafts cluster and localize at the IS in antigen-stimulated T cells. Here, we provide evidence that lipid raft polarization to the IS depends on an intracellular pathway that involves Vav1, Rac, and actin cytoskeleton reorganization. Thus, lipid rafts did not translocate to the IS in Vav1-deficient (Vav1-/-) T cells upon antigen stimulation. Similarly, T cell receptor transgenic Jurkat T cells also failed to translocate lipid rafts to the IS when transfected with dominant negative Vav1 mutants. Raft polarization induced by membrane-bound cholera toxin cross-linking was also abolished in Jurkat T cells expressing dominant negative Vav1 or Rac mutants and in cells treated with inhibitors of actin polymerization. However, Vav overexpression that induced F-actin polymerization failed to induce lipid rafts clustering. Therefore, Vav is necessary, but not sufficient, to regulate lipid rafts clustering and polarization at the IS, suggesting that additional signals are required.     

Lipid raft-dependent uptake, signalling and intracellular fate of Porphyromonas gingivalis in mouse macrophages

Lipid rafts are cholesterol-enriched microdomains involved in cellular trafficking and implicated as portals for certain pathogens. We sought to determine whether the oral pathogen Porphyromonas gingivalis enters macrophages via lipid rafts, and if so, to examine the impact of raft entry on its intracellular fate. Using J774A.1 mouse macrophages, we found that P. gingivalis colocalizes with lipid rafts in a cholesterol-dependent way. Depletion of cellular cholesterol using methyl-beta-cyclodextrin resulted in about 50% inhibition of P. gingivalis uptake, although this effect was reversed by cholesterol reconstitution. The intracellular survival of P. gingivalis was dramatically inhibited in cholesterol-depleted cells relative to untreated or cholesterol-reconstituted cells, even when infections were adjusted to allow equilibration of the initial intracellular bacterial load. P. gingivalis thus appeared to exploit raft-mediated uptake for promoting its survival. Consistent with this, lipid raft disruption enhanced the colocalization of internalized P. gingivalis with lysosomes. In contrast, raft disruption did not affect the expression of host receptors interacting with P. gingivalis, although it significantly inhibited signal transduction. In summary, P. gingivalis uses macrophage lipid rafts as signalling and entry platforms, which determine its intracellular fate to the pathogen's own advantage.     

Cutting edge: lipid raft integrity affects the efficiency of MHC class I tetramer binding and cell surface TCR arrangement on CD8+ T cells

Physically distinct cholesterol/sphingolipid-rich plasma membrane microdomains, so-called lipid rafts, have been recognized to play an important regulatory role in various cellular processes, from membrane trafficking to signal transduction, in a number of cell types. We report here that the ability of TCR on activated, functional CD8+ T lymphocytes to efficiently bind MHC class I tetramer complexes is dependent on the integrity of lipid rafts on the T lymphocyte membrane. We further provide evidence that TCR interact (associate) with lipid raft elements on the T cell surface before receptor engagement and that the topological arrangement of TCR on the cell surface is likewise influenced by lipid raft integrity.     

SCP-2/SCP-x gene ablation alters lipid raft domains in primary cultured mouse hepatocytes

Although reverse cholesterol transport from peripheral cell types is mediated through plasma membrane microdomains termed lipid rafts, almost nothing is known regarding the existence, protein/lipid composition, or structure of these putative domains in liver hepatocytes, cells responsible for the net removal of cholesterol from the body. Lipid rafts purified from hepatocyte plasma membranes by a nondetergent affinity chromatography method were: i) present at 33 +/- 3% of total plasma membrane protein; ii) enriched in key proteins of the reverse cholesterol pathway [scavenger receptor class B type I (SR-B1), ABCA1, P-glycoprotein (P-gp), sterol carrier protein-2 (SCP-2)]; iii) devoid of caveolin-1; iv) enriched in cholesterol, sphingomyelin, GM1, and phospholipids low in polyunsaturated fatty acid and double bond index; and v) exhibited an intermediate liquid-ordered lipid phase with significant transbilayer fluidity gradient. Ablation of the gene encoding SCP-2 significantly altered lipid rafts to: i) increase the proportion of lipid rafts present, thereby increasing raft total content of ABCA1, P-gp, and SR-B1; ii) increase total phospholipids while decreasing GM1 in lipid rafts; iii) decrease the fluidity of lipid rafts, consistent with the increased intermediate liquid-ordered phase; and iv) abolish the lipid raft transbilayer fluidity gradient. Thus, despite the absence of caveolin-1 in liver hepatocytes, lipid rafts represented nearly one-third of the mouse hepatocyte plasma membrane proteins and displayed unique protein, lipid, and biophysical properties that were differentially regulated by SCP-2 expression.     

Early and dynamic polarization of T cell membrane rafts and constituents prior to TCR stop signals

Polarization of membrane rafts and signaling proteins to form an immunological synapse is a hallmark of T cell stimulation. However, the kinetics of raft polarization and associated proteins in relation to the initial contact of the T cell with the APC are poorly defined. We addressed this question by measuring the distribution of membrane-targeted fluorescent protein markers during initial T cell interactions with B cell APCs. Experiments with unpulsed B cells lacking cognate Ag demonstrated an MHC class II-independent capping that was specific to membrane raft markers and required actin rearrangements and signals from Src kinases and PI3K. By live cell imaging experiments, we identified a similar specific polarization of membrane raft markers before TCR-dependent stop signals, and which occurred independently of cognate peptide-MHC class II. T cells conjugated to unpulsed B cells exhibited capping of CD4 and microclusters of the TCR zeta-chain, but only the CD4 enrichment was cholesterol dependent. Furthermore, raft association of CD4 was necessary for its efficient targeting to the Ag-independent caps. Interestingly, anergic Vbeta8(+) T cells isolated from staphylococcal enterotoxin B-injected mice did not exhibit Ag-independent capping of membrane rafts, showing that inhibition of these early, Ag-independent events is a property associated with tolerance. Altogether, these data show that membrane raft capping is one of the earliest events in T cell activation and represents one avenue for promoting and regulating downstream peptide-MHC-dependent signaling within the T cell.     

Regulation of CD1a surface expression and antigen presentation by invariant chain and lipid rafts

In immature dendritic cells (DCs), CD1a is almost exclusively expressed at the cell surface and its membrane organization is poorly understood. In this study, we report that MHC class II, invariant chain (Ii), and CD9 molecules are coimmunoprecipitated with CD1a in immature DCs, and that CD1a/Ii colocalization is dependent on lipid raft integrity. In HeLa-CIITA cells CD1a expression leads to increased Ii trafficking to the cell surface, confirming the relevance of this association. Furthermore, silencing of Ii in DCs induces significant CD1a accumulation on the plasma membrane whereas the total CD1a expression remains similar to that of control cells. These data suggest that CD1a recycling is facilitated by the association with the Ii. The CD1a localization in lipid rafts has functional relevance as demonstrated by inhibition of CD1a-restricted presentation following raft disruption. Overall, these findings identify Ii and lipid rafts as key regulators of CD1a organization on the surface of immature DCs and of its immunological function as Ag-presenting molecule.     

Ginsenoside Rh2 induces ligand-independent Fas activation via lipid raft disruption

Lipid rafts are plasma membrane platforms mediating signal transduction pathways for cellular proliferation, differentiation and apoptosis. Here, we show that membrane fluidity was increased in HeLa cells following treatment with ginsenoside Rh2 (Rh2), as determined by cell staining with carboxy-laurdan (C-laurdan), a two-photon dye designed for measuring membrane hydrophobicity. In the presence of Rh2, caveolin-1 appeared in non-raft fractions after sucrose gradient ultracentrifugation. In addition, caveolin-1 and GM1, lipid raft landmarkers, were internalized within cells after exposure to Rh2, indicating that Rh2 might disrupt lipid rafts. Since cholesterol overloading, which fortifies lipid rafts, prevented an increase in Rh2-induced membrane fluidity, caveolin-1 internalization and apoptosis, lipid rafts appear to be essential for Rh2-induced apoptosis. Moreover, Rh2-induced Fas oligomerization was abolished following cholesterol overloading, and Rh2-induced apoptosis was inhibited following treatment with siRNA for Fas. This result suggests that Rh2 is a novel lipid raft disruptor leading to Fas oligomerization and apoptosis.     

Human immunodeficiency virus type 1 Nef associates with lipid rafts to downmodulate cell surface CD4 and class I major histocompatibility complex expression and to increase viral infectivity

Lipid rafts are membrane microdomains that are functionally distinct from other membrane regions. We have shown that 10% of human immunodeficiency virus type 1 (HIV-1) Nef expressed in SupT1 cells is present in lipid rafts and that this represents virtually all of the membrane-associated Nef. To determine whether raft targeting, rather than simply membrane localization, has functional significance, we created a Nef fusion protein (LAT-Nef) containing the N-terminal 35 amino acids from LAT, a protein that is exclusively localized to rafts. Greater than 90% of the LAT-Nef protein was found in the raft fraction. In contrast, a mutated form, lacking two cysteine palmitoylation sites, showed less than 5% raft localization. Both proteins were equally expressed and targeted nearly exclusively to membranes. The LAT-Nef protein was more efficient than its nonraft mutant counterpart at downmodulating both cell surface CD4 and class I major histocompatibility complex (MHC) expression, as well as in enhancing first-round infectivity and being incorporated into virus particles. This demonstrates that targeting of Nef to lipid rafts is mechanistically important for all of these functions. Compared to wild-type Nef, LAT-Nef downmodulated class I MHC nearly as effectively as the wild-type Nef protein, but was only about 60% as effective for CD4 downmodulation and 30% as effective for infectivity enhancement. Since the LAT-Nef protein was found entirely in rafts while the wild-type Nef protein was distributed 10% in rafts and 90% in the soluble fraction, our results suggest that class I MHC downmodulation by Nef may be performed exclusively by raft-bound Nef. In contrast, CD4 downmodulation and infectivity enhancement may require a non-membrane-bound Nef component as well as the membrane-bound form.     

Aggregation of lipid rafts accompanies signaling via the T cell antigen receptor.

The role of lipid rafts in T cell antigen receptor (TCR) signaling was investigated using fluorescence microscopy. Lipid rafts labeled with cholera toxin B subunit (CT-B) and cross-linked into patches displayed characteristics of rafts isolated biochemically, including detergent resistance and colocalization with raft-associated proteins. LCK, LAT, and the TCR all colocalized with lipid patches, although TCR association was sensitive to nonionic detergent. Aggregation of the TCR by anti-CD3 mAb cross-linking also caused coaggregation of raft-associated proteins. However, the protein tyrosine phosphatase CD45 did not colocalize to either CT-B or CD3 patches. Cross-linking of either CD3 or CT-B strongly induced tyrosine phosphorylation and recruitment of a ZAP-70(SH2)(2)-green fluorescent protein (GFP) fusion protein to the lipid patches. Also, CT-B patching induced signaling events analagous to TCR stimulation, with the same dependence on expression of key TCR signaling molecules. Targeting of LCK to rafts was necessary for these events, as a nonraft- associated transmembrane LCK chimera, which did not colocalize with TCR patches, could not reconstitute CT-B-induced signaling. Thus, our results indicate a mechanism whereby TCR engagement promotes aggregation of lipid rafts, which facilitates colocalization of LCK, LAT, and the TCR whilst excluding CD45, thereby triggering protein tyrosine phosphorylation.     

Cholera toxin B-subunit prevents activation and proliferation of human CD4+ T cells by activation of a neutral sphingomyelinase in lipid rafts

The inhibition of human CD4+ T lymphocyte activation and proliferation by cholera toxin B-subunit (CTB) is a well-established phenomenon; nevertheless, the exact mechanism remained unclear. In the present study, we propose an explanation for the rCTB-induced inhibition of CD4+ T lymphocytes. rCTB specifically binds to GM1, a raft marker, and strongly modifies the lipid composition of rafts. First, rCTB inhibits sphingomyelin synthesis; second, it enhances phosphatidylcholine synthesis; and third, it activates a raft-resident neutral sphingomyelinase resembling to neutral sphingomyelinase type 1, thus generating a transient ceramide production. We demonstrated that these ceramides inhibit protein kinase Calpha phosphorylation and its translocation into the modified lipid rafts. Furthermore, we show that rCTB-induced ceramide production activate NF-kappaB. Combined all together: raft modification in terms of lipids, ceramide production, protein kinase Calpha inhibition, and NF-kappaB activation lead to CD4+ T cell inhibition.     

Protein interactions between CD2 and Lck are required for the lipid raft distribution of CD2

In T lymphocytes, lipid rafts are preferred sites for signal transduction initiation and amplification. Many cell membrane receptors, such as the TCR, coreceptors, and accessory molecules associate within these microdomains upon cell activation. However, it is still unclear in most cases whether these receptors interact with rafts through lipid-based amino acid modifications or whether raft insertion is driven by protein-protein interactions. In murine T cells, a significant fraction of CD2 associates with membrane lipid rafts. We have addressed the mechanisms that control the localization of rat CD2 at the plasma membrane, and its redistribution within lipid rafts induced upon activation. Following incubation of rat CD2-expressing cells with radioactive-labeled palmitic acid, or using CD2 mutants with Cys226 and Cys228 replaced by alanine residues, we found no evidence that rat CD2 was subjected to lipid modifications that could favor the translocation to lipid rafts, discarding palmitoylation as the principal mechanism for raft addressing. In contrast, using Jurkat cells expressing different CD2 and Lck mutants, we show that the association of CD2 with the rafts fully correlates with CD2 capacity to bind to Lck. As CD2 physically interacts with both Lck and Fyn, preferentially inside lipid rafts, and reflecting the increase of CD2 in lipid rafts following activation, CD2 can mediate the interaction between the two kinases and the consequent boost in kinase activity in lipid rafts.     

Distinct MHC Class II Molecules Are Associated on the Dendritic Cell Surface in Cholesterol-dependent Membrane Microdomains

Very small amounts of MHC class II-peptide complexes expressed on the surface of antigen-presenting cells (APCs) are capable of stimulating antigen-specific CD4 T cells. There is intense interest to elucidate the molecular mechanisms by which these small amounts of MHC-II can cluster, cross-link T cell receptors, and promote T cell proliferation. We now demonstrate that a significant fraction of the total pool of MHC-II molecules on the surface of dendritic cells is physically associated in macromolecular aggregates. These MHC-II/MHC-II interactions have been probed by co-immunoprecipitation analysis of the MHC-II I-A molecule with the related I-E molecule. These molecular associations are maintained in gentle detergents but are disrupted in harsh detergents such as Triton X-100. MHC-II I-A/I-E interactions are disrupted when plasma membrane cholesterol is extracted using methyl β-cyclodextrin, suggesting that lipid raft microdomains are important mediators of these MHC-II interactions. Although it has been proposed that tetraspanin proteins regulate molecular clustering, aggregation, and co-immunoprecipitation in APCs, genetic deletion of the tetraspanin family members CD9 or CD81 had no effect on MHC-II I-A/I-E binding. These data demonstrate that the presence of distinct forms of MHC-II with plasma membrane lipid rafts is required for MHC-II aggregation in APCs and provides a molecular mechanism allowing dendritic cells expressing small amounts of MHC-II-peptide complexes to cross-link and stimulate CD4 T cells.