Selective inhibition of T cell activation via CD147 through novel modulation of lipid rafts

The plasma membrane is compartmentalized into microdomains and the association/dissociation of receptors and signaling molecules with/from these membrane domains is a major principle for regulation of signal transduction. By following the reorganization of microdomains on living cells and performing biochemical studies, we show that Ab targeting of the T cell activation-associated Ag CD147 prevents TCR stimulation-dependent reorganization and clustering of microdomains. Triggering CD147 induces a displacement of the GPI-anchored coreceptors CD48 and CD59 from microdomains in human T lymphocytes. This perturbation of microdomains is accompanied by a selective inhibition of TCR-mediated T cell proliferation. The CD147-inhibited cells secret normal levels of IL-2 but acquire reduced amounts of the IL-2 receptor alpha-chain CD25. These results indicate that negative regulating signals can modulate microdomains and suggest a general mechanism for inhibition of receptor signaling.     

Regulated recruitment of MHC class II and costimulatory molecules to lipid rafts in dendritic cells

T cell activation has long been associated with the partitioning of Ag receptors and associated molecules to lipid microdomains. We now show that dendritic cells (DCs) also accomplish the selective recruitment to lipid rafts of molecules critical for Ag presentation. Using mouse bone marrow-derived DCs, we demonstrate that MHC class II molecules become substantially localized to rafts upon DC maturation. Even more striking is the fact that CD86 is recruited to rafts upon T cell-DC interaction. Recruitment is Ag dependent and requires CD28 on T cells. Despite the regulated recruitment of MHC class II and CD86 to rafts, unlike the counter-receptors in T cells, DCs do not polarize these molecules to sites of DC-T cell contact. This difference may reflect the necessity for DCs to interact with multiple T cells simultaneously and emphasizes that the biochemical and morphological correlates of lipid rafts are not necessarily equivalent.     

TCR- and CD28-mediated recruitment of phosphodiesterase 4 to lipid rafts potentiates TCR signaling

Ligation of the TCR along with the coreceptor CD28 is necessary to elicit T cell activation in vivo, whereas TCR triggering alone does not allow a full T cell response. Upon T cell activation of human peripheral blood T cells, we found that the majority of cAMP was generated in T cell lipid rafts followed by activation of protein kinase A. However, upon TCR and CD28 coligation, beta-arrestin in complex with cAMP-specific phosphodiesterase 4 (PDE4) was recruited to lipid rafts which down-regulated cAMP levels. Whereas inhibition of protein kinase A increased TCR-induced immune responses, inhibition of PDE4 blunted T cell cytokine production. Conversely, overexpression of either PDE4 or beta-arrestin augmented TCR/CD28-stimulated cytokine production. We show here for the first time that the T cell immune response is potentiated by TCR/CD28-mediated recruitment of PDE4 to lipid rafts, which counteracts the local, TCR-induced production of cAMP. The specific recruitment of PDE4 thus serves to abrogate the negative feedback by cAMP which is elicited in the absence of a coreceptor stimulus.     

Lipid-dependent recruitment of neuronal Src to lipid rafts in the brain

Although most Src family tyrosine kinases are modified by palmitoylation as well as myristoylation, Src itself is only myristoylated. Dual acylation is important for attachment to liquid-ordered microdomains or lipid rafts. Accordingly, Src is excluded from lipid rafts in fibroblasts. Evidence of partial genetic redundancy between Src and Fyn for brain-specific targets suggests that these two kinases may occupy overlapping subcellular locations. Neuronal Src (NSrc), an alternative isoform of Src with a 6-amino acid insert in the Src homology 3 domain, is highly expressed in neurons. We investigated whether this structural difference in NSrc allows it to associate with lipid rafts. We found that perinatal mouse brains express predominantly NSrc, which is partly (10-20%) in a lipid raft fraction from brain but not fibroblasts. The association of Src with brain lipid rafts does not depend on the NSrc insert but depends on the amino-terminal myristoylation signal. A crude lipid fraction from brain promotes NSrc entry into rafts in vitro. Moreover, lipid raft-localized NSrc is more catalytically active than NSrc from the soluble fraction, possibly because raft localization alters access to other tyrosine kinases and phosphatases. These findings suggest that NSrc may be involved in signaling from lipid rafts in mouse brain.     

Phagocytic signaling molecules in lipid rafts of COS-1 cells transfected with FcgammaRIIA

COS-1 cells bearing FcgammaRIIA were used as a model to demonstrate co-localization of several enzymes previously shown to regulate neutrophil phagocytosis. In COS-1 cells, phospholipase D (PLD) in the membrane fraction was activated during phagocytosis. PLD was found almost exclusively in lipid rafts, along with RhoA and ARF1. Protein kinase C-delta (PKCdelta) and Raf-1 translocated to lipid rafts. In neutrophils, ceramide levels increase during phagocytosis, indicating that FcgammaRIIA engagement initiates ceramide generation. Applying this model, we transfected COS-1 cells with FcgammaRIIA that had been mutated in the ITAM region, rendering them unable to ingest particles. When the mutant receptors were engaged, ceramide was generated and MAPK was activated normally, thus these processes did not require actual ingestion of particles. These results indicate that signaling proteins for phagocytosis are either constitutively present in, or are recruited to, lipid rafts where they are readily available to activate one another.     

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.     

Differential recruitment of Kv1.4 and Kv4.2 to lipid rafts by PSD-95

The activity of voltage-gated potassium (Kv) channels, and consequently their influence on cellular functions, can be substantially altered by phosphorylation. Several protein kinases that modulate Kv channel activity are found in membrane subdomains known as lipid rafts, which are thought to organize signaling complexes in the cell. Thus, we asked whether Kv1.4 and Kv4.2, two channels with critical roles in excitable cells, are found in lipid rafts. Acylation can target proteins to raft regions; however, Kv channels are not acylated, and therefore, a different mechanism must exist to bring them into these membrane subdomains. Because both Kv1.4 and Kv4.2 interact with postsynaptic density protein 95 (PSD-95), which is acylated (specifically, palmitoylated), we examined whether PSD-95 can recruit these channels to lipid rafts. We found that a portion of Kv1.4 and Kv4.2 protein in rat brain membranes is raft-associated. Lipid raft patching and immunostaining confirmed that some Kv4.2 is in Thy-1-containing rafts in rat hippocampal neurons. Using a heterologous expression system, we determined that palmitoylation of PSD-95 was crucial to its localization to lipid rafts. We then assessed the contribution of PSD-95 to the raft association of these channels. Co-expression of PSD-95 increased the amount of Kv1.4, but not Kv4.2, in lipid rafts. Deleting the PSD-95 binding motif of Kv1.4 eliminated this recruitment, as did substituting a palmitoylation-deficient PSD-95 mutant. This work represents the first evidence that PSD-95 binding can recruit Kv channels into lipid rafts, a process that could facilitate interactions with the protein kinases that affect channel activity.     

LAT displacement from lipid rafts as a molecular mechanism for the inhibition of T cell signaling by polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs) suppress immune responses and inhibit T cell activation through largely unknown mechanisms. The displacement of signaling proteins from membrane lipid rafts has recently been suggested as underlying PUFA-mediated T cell inhibition. We show here that PUFA treatment specifically interferes with T cell signal transduction by blocking tyrosine phosphorylation of LAT (linker for activation of T cells) and phospholipase Cgamma1. A significant fraction of LAT was displaced from rafts by PUFA treatment along with other signaling proteins. However, retaining LAT alone in lipid rafts effectively restored phospholipase Cgamma1/calcium signaling in PUFA-treated T cells. These data reveal LAT displacement from lipid rafts as a molecular mechanism by which PUFAs inhibit T cell signaling and underline the predominant importance of LAT localization in rafts for efficient T cell activation.     

Activated ezrin promotes cell migration through recruitment of the GEF Dbl to lipid rafts and preferential downstream activation of Cdc42

Establishment of polarized cell morphology is a critical factor for migration and requires precise spatial and temporal activation of the Rho GTPases. Here, we describe a novel role of the actin-binding ezrin/radixin/moesin (ERM)-protein ezrin to be involved in recruiting Cdc42, but not Rac1, to lipid raft microdomains, as well as the subsequent activation of this Rho GTPase and the downstream effector p21-activated kinase (PAK)1, as shown by fluorescence lifetime imaging microscopy. The establishment of a leading plasma membrane and the polarized morphology necessary for random migration are also dependent on ERM function and Cdc42 in motile breast carcinoma cells. Mechanistically, we show that the recruitment of the ERM-interacting Rho/Cdc42-specific guanine nucleotide exchange factor Dbl to the plasma membrane and to lipid raft microdomains requires the phosphorylated, active conformer of ezrin, which serves to tether the plasma membrane or its subdomains to the cytoskeleton. Together these data suggest a mechanism whereby precise spatial guanine nucleotide exchange of Cdc42 by Dbl is dependent on functional ERM proteins and is important for directional cell migration.     

Released GFRalpha1 potentiates downstream signaling, neuronal survival, and differentiation via a novel mechanism of recruitment of c-Ret to lipid rafts

Although both c-Ret and GFRalpha1 are required for responsiveness to GDNF, GFRalpha1 is widely expressed in the absence of c-Ret, suggesting alternative roles for "ectopic" sites of GFRalpha1 expression. We show that GFRalpha1 is released by neuronal cells, Schwann cells, and injured sciatic nerve. c-Ret stimulation in trans by soluble or immobilized GFRalpha1 potentiates downstream signaling, neurite outgrowth, and neuronal survival, and elicits dramatic localized expansions of axons and growth cones. Soluble GFRalpha1 mediates robust recruitment of c-Ret to lipid rafts via a novel mechanism requiring the c-Ret tyrosine kinase. Activated c-Ret associates with different adaptor proteins inside and outside lipid rafts. These results provide an explanation for the tissue distribution of GFRalpha1, supporting the physiological importance of c-Ret activation in trans as a novel mechanism to potentiate and diversify the biological responses to GDNF.     

Dynamic recruitment of human CD2 into lipid rafts. Linkage to T cell signal transduction

CD2 mediates T cell adhesion via its ectodomain and signal transduction utilizing its 117-amino acid cytoplasmic tail. Here we show that a significant fraction of human CD2 molecules is inducibly recruited into lipid rafts upon CD2 cross-linking by a specific pair of mitogenic anti-CD2 monoclonal antibodies (anti-T11(2) + anti-T11(3)) or during cellular conjugate formation by CD58, the physiologic ligand expressed on antigen-presenting cells. Translocation to lipid microdomains is independent of the T cell receptor (TCR) and, unlike inducible TCR-raft association, requires no tyrosine phosphorylation. Structural integrity of rafts is necessary for CD2-stimulated elevation of intracellular free calcium and tyrosine phosphorylation of cellular substrates. Whereas murine CD2 contains two membrane-proximal intracellular cysteines, partitioning CD2 into cholesterol-rich lipid rafts constitutively, human CD2 has no cytoplasmic cysteines. Mapping studies using CD2 point mutation, deletion, and chimeric molecules suggest that conformational change in the CD2 ectodomain participates in inducible raft association and excludes the membrane-proximal N-linked glycans, the transmembrane segment, and the CD2 cytoplasmic region (residues 8-117) as necessary for translocation. Translocation of CD2 into lipid rafts may reorganize the membrane into an activation-ready state prior to TCR engagement by a peptide associated with a major histocompatibility complex molecule, accounting for synergistic T cell stimulation by CD2 and the TCR.     

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.     

Biological activity of neurotrophins is dependent on recruitment of Rac1 to lipid rafts

The Rho family of small GTPases, key regulators of the actin cytoskeleton in eukaryotic cells, is implicated in the control of neuronal morphology. Here, we report that neurotrophin dependent cytoskeletal changes, characteristic of the phenotype of Rac1, in the hippocampal neurons or PC12 cells are inhibited by the disruption of lipid raft integrity. Activation of Rac1 induced by NGF is impaired in cholesterol-depleted PC12 cells. Pretreatment with gammaGTP shifted significant amount of Rac1, presumably in a GTP-bound form, from non-raft to raft fractions. Proper recruitment of activated Rac1 to lipid rafts, structures that represent specialized signaling organelles, is of fundamental importance in determining neurotrophins' bioactivity.     

Proteome analysis of lipid rafts in Jurkat cells characterizes a raft subset that is involved in NF-kappaB activation

Lipid rafts are detergent-insoluble membrane domains that play a key role in signal transduction by the T-cell antigen receptor. Proteome analysis revealed the presence of amidosulfobetaine-soluble signal transducing, integral membrane, cytoskeletal, heat shock, and GTP-binding proteins in rafts prepared from Jurkat cells. Several of these proteins were recruited to rafts by CD3/CD28 costimulation. Of particular interest is the inducible association of activated IkappaB kinase complexes with raft vesicles that could be captured with anti-flotillin-1 antibodies. Following amidosulfobetaine solubilization, flotillin-beta and IKKbeta underwent reciprocal co-immunoprecipitation. Treatment of Jurkat cells with methyl-beta-cyclodextrin disrupted the assembly and activation of this raft complex and also interfered in CD3/ CD28-induced activation of a NF-kappaB response element in the IL-2 promoter.     

Segregation of Nogo66 receptors into lipid rafts in rat brain and inhibition of Nogo66 signaling by cholesterol depletion

NogoA, a myelin-associated component, inhibits neurite outgrowth. Nogo66, a portion of NogoA, binds to Nogo66 receptor (NgR) and induces the inhibitory signaling. LINGO-1 and p75 neurotrophin receptor (p75), the low-affinity nerve growth factor receptor, are also required for NogoA signaling. However, signaling mechanisms downstream to Nogo receptor remain poorly understood. Here, we observed that NgR and p75 were colocalized in low-density membrane raft fractions derived from forebrains and cerebella as well as from cerebellar granule cells. NgR interacted with p75 in lipid rafts. In addition, disruption of lipid rafts by beta-methylcyclodextrin, a cholesterol-binding reagent, reduced the Nogo66 signaling. Our results suggest an important role of lipid rafts in facilitating the interaction between NgRs and provide insight into mechanisms underlying the inhibition of neurite outgrowth by NogoA.     

T-cell antigen receptor triggering and lipid rafts: a matter of space and time scales. Talking Point on the involvement of lipid rafts in T-cell activation

T-cell antigen receptor triggering mechanisms and lipid rafts are of broad interest, but are also controversial topics. Here, we review some recent progress in these two research fields, which has been accomplished mostly in live cells and with the use of advanced technologies. We then discuss the potential relationship between membrane-domain organization and T-cell antigen receptor-triggering mechanisms. On the basis of the relevant experimental observations, we argue that the key to achieving a better understanding of both processes is the ability to monitor the molecular dynamics and interactions taking place in the membrane of T cells at a spatial scale of tens to hundreds of nanometres, with a subsecond-to-second temporal resolution.     

G(s) signaling is intact after disruption of lipid rafts.

Membrane microdomains enriched in cholesterol and sphingolipids modulate a number of signal transduction pathways and provide a residence for heterotrimeric G proteins, their receptors, and their effectors. We investigated whether signaling through G(s) was dependent on these membrane domains, characterized by their resistance to detergents, by depleting cells of cholesterol and sphingolipids. For cholesterol depletion, rat salivary epithelial A5 cells were cultured under low-cholesterol conditions, and then treated with the cholesterol chelator methyl-beta-cyclodextrin. For sphingolipid depletion, LY-B cells, a mutant CHO cell line that is unable to synthesize sphingolipids, were incubated under low-sphingolipid conditions. Depletion of cholesterol or sphingolipid led to a loss or decrease, respectively, in the amount of Galpha(s) from the detergent-resistant membranes without any change in the cellular or membrane-bound amounts of Galpha(s). The cAMP accumulation in response to a receptor agonist was intact and the level slightly increased in cells depleted of cholesterol or sphingolipids compared to that in control cells. These results indicate that localization of Galpha(s) to detergent-resistant membranes was not required for G(s) signaling. Analysis of the role of lipid rafts on the kinetics of protein associations in the membrane suggests that compartmentalization in lipid rafts may be more effective in inhibiting protein interactions and, depending on the pathway, ultimately inhibit or promote signaling.     

Specific inhibition of the antibody-mediated activation of a defective beta-D-galactosidase by circulating activating epitope-binding molecules.

Activation of a defective Escherichia coli beta-D-galactosidase by specific activating antibody is inhibited competitively by a molecule with immunoglobulin properties but devoid of activating capacity. This molecule is found in the serum of nonimmunized rabbits and is no longer detectable after beta-D-galactosidase administration, but can be demonstrated in rabbits injected with antigens other than the enzyme. The data show that the inhibitory molecule recognizes and interacts specifically with the activating epitope of the activatable enzyme and that, although unable to activate the latter, it competes with the activating antibody and inhibits activation.     

Spatial memory formation induces recruitment of NMDA receptor and PSD-95 to synaptic lipid rafts

NMDA receptors (NMDARs) activation in the hippocampus and insular cortex is necessary for spatial memory formation. Recent studies suggest that localization of NMDARs to lipid rafts enhance their signalization, since the kinases that phosphorylate its subunits are present in larger proportion in lipid raft membrane microdomains. We sought to determine the possibility that NMDAR translocation to synaptic lipid rafts occurs during plasticity processes such as memory formation. Our results show that water maze training induces a rapid recruitment of NMDAR subunits (NR1, NR2A, NR2B) and PSD-95 to synaptic lipid rafts and decrease in the post-synaptic density plus an increase of NR2B phosphorylation at tyrosine 1472 in the rat insular cortex. In the hippocampus, spatial training induces selective translocation of NR1 and NR2A subunits to lipid rafts. These results suggest that NMDARs translocate from the soluble fraction of post-synaptic membrane (non-raft PSD) to synaptic lipid raft during spatial memory formation. The recruitment of NMDA receptors and other proteins to lipid rafts could be an important mechanism for increasing the efficiency of synaptic transmission during synaptic plasticity process.