Lipid rafts regulate cellular CD40 receptor localization in vascular endothelial cells

Cholesterol enriched lipid rafts are considered to function as platforms involved in the regulation of membrane receptor signaling complex through the clustering of signaling molecules. In this study, we tested whether these specialized membrane microdomains affect CD40 localization in vitro and in vivo. Here, we provide evidence that upon CD40 ligand stimulation, endogenous and exogenous CD40 receptor is rapidly mobilized into lipid rafts compared with unstimulated HAECs. Efficient binding between CD40L and CD40 receptor also increases amounts of CD40 protein levels in lipid rafts. Deficiency of intracellular conserved C terminus of the CD40 cytoplasmic tail impairs CD40 partitioning in raft. Raft disorganization after methyl-beta-cyclodextrin treatment diminishes CD40 localization into rafts. In vivo studies show that elevation of circulating cholesterol in high-cholesterol fed rabbits increases the cholesterol content and CD40 receptor localization in lipid rafts. These findings identify a physiological role for membrane lipid rafts as a critical regulator of CD40-mediated signal transduction and raise the possibility that certain pathologic conditions may be treated by altering CD40 signaling with drugs affecting its raft localization.     

Compartmentalized DCC signalling is distinct from DCC localized to lipid rafts

Background information. Netrin‐1 is a bi‐functional cue that attracts or repels different classes of neurons during development. The netrin‐1 receptor DCC (deleted in colorectal cancer) acts as a tyrosine kinase‐associated receptor to mediate the attractive response towards netrin‐1. The lipid raft‐localized Src family kinase Fyn is required for DCC‐mediated axon guidance. DCC functions are also dependent on lipid rafts, membrane microdomains corresponding to a low‐density, detergent‐resistant membrane fraction. However, it remains unclear how the association of DCC with lipid rafts controls netrin‐1 signalling. Results. DCC targeted to lipid rafts represented a minor proportion of total DCC inside the cell, but predominated on the cell surface of both IMR‐32 human neuroblastoma cells and embryonic cortical neurons. Netrin‐1 accumulated in lipid rafts, but had no effect on the targeting of DCC to that compartment, with DCC remaining on the cell surface in lipid rafts through 60 min post‐treatment. However, DCC was able to interact with Fyn, both in the lipid rafts and soluble compartments isolated from embryonic E19 rat brains, whereas early downstream signalling components such as Nck‐1, and total and active focal adhesion kinase were mainly localized to the non‐lipid raft compartment. Conclusions. Together, these results suggest that DCC can be found in raft and non‐raft portions of the plasma membrane, with early signalling events propagated by non‐raft associated DCC.     

Lipid rafts in neuregulin signaling at synapses

Neuregulins are a family of EGF domain-containing factors that play an important role in development. In the nervous system, they promote glial differentiation, induce neurotransmitter receptor expression, and regulate synaptic plasticity. Recent studies indicate that ErbB protein tyrosine kinases, neuregulin receptors, translocate to lipid raft microdomains in the plasma membrane in response to neuregulin. Localization of ErbB proteins in lipid rafts appeared to be necessary for neuregulin signaling and regulation of synaptic plasticity. We will review recent studies of lipid rafts and neuregulin function and discuss possible roles of lipid rafts in compartmentalized neuregulin signaling and translocation of ErbB proteins to synapses.     

Ethanol mimics ligand-mediated activation and endocytosis of IL-1RI/TLR4 receptors via lipid rafts caveolae in astroglial cells

We have recently reported that ethanol-induced inflammatory processes in the brain and glial cells are mediated via the activation of interleukin-1 beta receptor type I (IL-1RI)/toll-like receptor type 4 (TLR4) signalling. The mechanism(s) by which ethanol activates these receptors in astroglial cells remains unknown. Recently, plasma membrane microdomains, lipid rafts, have been identified as platforms for receptor signalling and, in astrocytes, rafts /caveolae constitute an important integrators of signal events and trafficking. Here we show that stimulation of astrocytes with IL-1β, lipopolysaccharide or ethanol (10 and 50 mM), triggers the translocation of IL-1RI and/or TLR4 into lipid rafts caveolae-enriched fractions, promoting the recruitment of signalling molecules (phospho-IL-1R-associated kinase and phospho-extracellular regulated-kinase) into these microdomains. With confocal microscopy, we further demonstrate that IL-1RI is internalized by caveolar endocytosis via enlarged caveosomes organelles upon IL-1β or ethanol treatment, which sorted their IL-1RI cargo into the endoplasmic reticulum–Golgi compartment and into the nucleus of astrocytes. In short, our findings demonstrate that rafts /caveolae are critical for IL-1RI and TLR4 signalling in astrocytes, and reveal a novel mechanism by which ethanol, by interacting with lipid rafts caveolae, promotes IL-1RI and TLR4 receptors recruitment, triggering their endocytosis via caveosomes and downstream signalling stimulation. These results suggest that TLRs receptors are important targets of ethanol-induced inflammatory damage in the brain.     

Lipid rafts have different sizes depending on membrane composition: a time-resolved fluorescence resonance energy transfer study

The ternary lipid system palmitoylsphingomyelin (PSM)/palmitoyloleoylphosphatidylcholine (POPC)/cholesterol is a model for lipid rafts. Previously the phase diagram for that mixture was obtained, establishing the composition and boundaries for lipid rafts. In the present work, this system is further studied in order to characterize the size of the rafts. For this purpose, a time-resolved fluorescence resonance energy transfer (FRET) methodology, previously applied with success to a well-characterized phosphatidylcholine/cholesterol binary system, is used. It is concluded that: (1) the rafts on the low raft fraction of the raft region are small (below 20 nm), whereas on the other side the domains are larger; (2) on the large domain region, the domains reach larger sizes in the ternary system (> approximately 75-100 nm) than in binary systems phosphatidylcholine/cholesterol (between approximately 20 and approximately 75-100 nm); (3) the raft marker ganglioside G(M1) in small amounts (and excess cholera toxin subunit B) does not affect the general phase behaviour of the lipid system, but can increase the size of the rafts on the small to intermediate domain region. In summary, lipid-lipid interactions alone can originate lipid rafts on very different length scales. The conclusions presented here are consistent with the literature concerning both model systems and cell membrane studies.     

Role of lipid rafts in agrin-elicited acetylcholine receptor clustering

Emerging concepts of membrane organization point to the compartmentalization of the plasma membrane into distinct lipid microdomains. This lateral segregation within cellular membranes is based on cholesterol-sphingolipid-enriched microdomains or lipid rafts which can move laterally and assemble into large-scale domains to create plasma membrane specialized cellular structures at specific cell locations. Such domains are likely involved in the genesis of the postsynaptic specialization at the neuromuscular junction, which requires the accumulation of acetylcholine receptors (AChRs), through activation of the muscle specific kinase MuSK by the neurotropic factor agrin and the reorganization of the actin cytoskeleton. We used C2C12 myotubes as a model system to investigate whether agrin-elicited AChR clustering correlated with lipid rafts. In a previous study, using two-photon Laurdan confocal imaging, we showed that agrin-induced AChR clusters corresponded to condensed membrane domains: the biophysical hallmark of lipid rafts [F. Stetzkowski-Marden, K. Gaus, M. Recouvreur, A. Cartaud, J. Cartaud, Agrin elicits membrane condensation at sites of acetylcholine receptor clusters in C2C12 myotubes, J. Lipid Res. 47 (2006) 2121-2133]. We further demonstrated that formation and stability of AChR clusters depend on cholesterol. We also reported that three different extraction procedures (Triton X-100, pH 11 or isotonic Ca++, Mg++ buffer) generated detergent resistant membranes (DRMs) with similar cholesterol/GM1 ganglioside content, which are enriched in several signalling postsynaptic components, notably AChR, the agrin receptor MuSK, rapsyn and syntrophin. Upon agrin engagement, actin and actin-nucleation factors such as Arp2/3 and N-WASP were transiently recovered within raft fractions suggesting that the activation by agrin can trigger actin polymerization. Taken together, the present data suggest that AChR clustering at the neuromuscular junction relies upon a mechanism of raft coalescence driven by agrin-elicited actin polymerization.     

A novel approach to examining compositional heterogeneity of detergent-resistant lipid rafts

Lipid rafts play an important role in cell signalling, cell adhesion and other cellular functions. Compositional heterogeneity of lipid rafts provides one mechanism of how lipid rafts provide the spatial and temporal regulation of cell signalling and cell adhesion. The constitutive presence of some signalling receptors/molecules and accumulation of others in the lipid raft allows them to interact with each other and thereby facilitate relay of signals from the plasma membrane to the cell interior. Devising a method that can analyze these lipid microdomains for the presence of signalling receptors/molecules on an individual raft basis is required to address the issue of lipid raft heterogeneity. SDS-PAGE analysis, currently used for analyses of detergent-resistant lipid rafts, does not address this question. We have designed a cell-free assay that captures detergent-resistant lipid rafts with an antibody against a raft-resident molecule and detects the presence of another lipid raft molecule. Our results suggest that detergent-resistant lipid rafts, also known as detergent-resistant membranes, are heterogeneous populations on an immortalized mouse T-cell plasma membrane with respect to antigen receptor/signalling complex and other signalling/adhesion proteins. This cell-free assay provides a simple and quick way to examine the simultaneous presence of two proteins in the lipid rafts and has the potential to estimate trafficking of molecules in and out of the lipid microdomains during cell signalling on a single detergent-resistant lipid raft basis.     

Proteome analysis of adipocyte lipid rafts reveals that gC1qR plays essential roles in adipogenesis and insulin signal transduction

Since insulin receptors and their downstream signaling molecules are organized in lipid rafts, proteomic analysis of adipocyte lipid rafts may provide new insights into the function of lipid rafts in adipogenesis and insulin signaling. To search for proteins involved in adipocyte differentiation and insulin signaling, we analyzed detergent‐resistant lipid raft proteins from 3T3‐L1 preadipocytes and adipocytes by 2‐DE. Eleven raft proteins were identified from adipocytes. One of the adipocyte‐specific proteins was globular C1q receptor (gC1qR), an acidic 32 kDa protein known as the receptor for the globular domain of complement C1q. The targeting of gC1qR into lipid rafts was significantly increased during adipogenesis, as determined by immunoblotting and immunofluorescence. Since the silencing of gC1qR by small RNA interference abolished adipogenesis and blocked insulin‐induced activation of insulin receptor, insulin receptor substrate‐1 (IRS‐1), Akt, and Erk1/2, we can conclude that gC1qR is an essential molecule involved in adipogenesis and insulin signaling.     

Lipid rafts serve as signaling platforms for Tie2 receptor tyrosine kinase in vascular endothelial cells

The Tie2 receptor tyrosine kinase plays a pivotal role in vascular and hematopoietic development. The major intracellular signaling systems activated by Tie2 in response to Angiopoietin-1 (Ang1) include the Akt and Erk1/2 pathways. Here, we investigated the role of cholesterol-rich plasma membrane microdomains (lipid rafts) in Tie2 regulation. Tie2 could not be detected in the lipid raft fraction of human umbilical vein endothelial cells (HUVECs) unless they were first stimulated with Ang1. After stimulation, a minor fraction of Tie2 associated tightly with the lipid rafts. Treatment of HUVECs with the lipid raft disrupting agent methyl-β-cyclodextrin selectively inhibited Ang1-induced Akt phosphorylation, but not Erk1/2 phosphorylation. It has been reported that inhibition of FoxO activity is an important mechanism for Ang1-stimulated Tie2-mediated endothelial function. Consistent with this, we found that phosphorylation of FoxO mediated by Tie2 activation was attenuated by lipid raft disruption. Therefore, we propose that lipid rafts serve as signaling platforms for Tie2 receptor tyrosine kinase in vascular endothelial cells, especially for the Akt pathway.     

Analysis of CD44-containing lipid rafts: Recruitment of annexin II and stabilization by the actin cytoskeleton.

CD44, the major cell surface receptor for hyaluronic acid (HA), was shown to localize to detergent-resistant cholesterol-rich microdomains, called lipid rafts, in fibroblasts and blood cells. Here, we have investigated the molecular environment of CD44 within the plane of the basolateral membrane of polarized mammary epithelial cells. We show that CD44 partitions into lipid rafts that contain annexin II at their cytoplasmic face. Both CD44 and annexin II were released from these lipid rafts by sequestration of plasma membrane cholesterol. Partition of annexin II and CD44 to the same type of lipid rafts was demonstrated by cross-linking experiments in living cells. First, when CD44 was clustered at the cell surface by anti-CD44 antibodies, annexin II was recruited into the cytoplasmic leaflet of CD44 clusters. Second, the formation of intracellular, submembranous annexin II-p11 aggregates caused by expression of a trans-dominant mutant of annexin II resulted in coclustering of CD44. Moreover, a frequent redirection of actin bundles to these clusters was observed. These basolateral CD44/annexin II-lipid raft complexes were stabilized by addition of GTPgammaS or phalloidin in a semipermeabilized and cholesterol-depleted cell system. The low lateral mobility of CD44 in the plasma membrane, as assessed with fluorescent recovery after photobleaching (FRAP), was dependent on the presence of plasma membrane cholesterol and an intact actin cytoskeleton. Disruption of the actin cytoskeleton dramatically increased the fraction of CD44 which could be recovered from the light detergent-insoluble membrane fraction. Taken together, our data indicate that in mammary epithelial cells the vast majority of CD44 interacts with annexin II in lipid rafts in a cholesterol-dependent manner. These CD44-containing lipid microdomains interact with the underlying actin cytoskeleton.     

The platelet receptor for type III collagen (TIIICBP) is present in platelet membrane lipid microdomains (rafts)

Platelet interactions with collagen are orchestrated by the presence or the migration of platelet receptor(s) for collagen into lipid rafts, which are specialized lipid microdomains from the platelet plasma membrane enriched in signalling proteins. Electron microscopy shows that in resting platelets, TIIICBP, a receptor specific for type III collagen, is present on the platelet membrane and associated with the open canalicular system, and redistributes to the platelet membrane upon platelet activation. After platelet lysis by 1% Triton X-100 and the separation of lipid rafts on a discontinuous sucrose gradient, TIIICBP is recovered in lipid raft-containing fractions and Triton X-100 insoluble fractions enriched in cytoskeleton proteins. Platelet aggregation, induced by type III collagen, was inhibited after disruption of the lipid rafts by cholesterol depletion, whereas platelet adhesion under static conditions did not require lipid raft integrity. These results indicate that TIIICBP, a platelet receptor involved in platelet interaction with type III collagen, is localized within platelet lipid rafts where it could interact with other platelet receptors for collagen (GP VI and α2β1 integrin) for efficient platelet activation. Pascal Maurice and Ludovic Waeckel have contributed equally to this work.     

The γ-aminobutyric acid receptor B, but not the metabotropic glutamate receptor type-1, associates with lipid rafts in the rat cerebellum

Recent evidence suggests that specialized microdomains, called lipid rafts, exist within plasma membranes. These domains are enriched in cholesterol and sphingolipids and are resistant to non-ionic detergent-extraction at 4 degrees C. They contain specific populations of membrane proteins, and can change their size and composition in response to cellular signals, resulting in activation of signalling cascades. Here, we demonstrate that both the metabotropic gamma-aminobutyric acid receptor B (GABA(B) receptor) and the metabotropic glutamate receptor-1 from rat cerebellum are insoluble in the non-ionic detergent Triton X-100. However, only the GABA(B) receptor associates with raft fractions isolated from rat brain by sucrose gradient centrifugation. Moreover, increasing the stringency of isolation by decreasing the protein : detergent ratio caused an enrichment of the GABA(B) receptor in raft fractions. In contrast, depletion of cholesterol from cerebellar membranes by either saponin or methyl-beta-cyclodextrin treatment, which solubilize known raft markers, also increased the solubility of the GABA(B) receptor. These properties are all consistent with an association of the GABA(B) receptor with lipid raft microdomains.     

Transient translocation of the B cell receptor and Src homology 2 domain-containing inositol phosphatase to lipid rafts: evidence toward a role in calcium regulation

Membrane microdomains (lipid rafts) are enriched in selected signaling molecules and may compartmentalize receptor-mediated signals. Here, we report that in primary human B lymphocytes and in Ramos B cells B cell receptor (BCR) stimulation induces rapid and transient redistribution of a subset of engaged BCRs to lipid rafts and phosphorylation of raft-associated tyrosine kinase substrates. Cholesterol sequestration disrupted the lipid rafts, preventing BCR redistribution, but did not inhibit tyrosine kinase activation or phosphorylation of mitogen-activated protein kinase/extracellular regulated kinase. However, raft disruption enhanced the release of calcium from intracellular stores, suggesting that rafts may sequester early signaling events that down-regulate calcium flux. Consistent with this, BCR stimulation induced rapid and transient translocation of the Src homology 2 domain-containing inositol phosphatase, SHIP, into lipid rafts.     

The use of styrene-maleic acid copolymer (SMA) for studies on T cell membrane rafts

An emerging alternative to the use of detergents in biochemical studies on membrane proteins is apparently the use styrene-maleic acid (SMA) amphipathic copolymers. These cut the membrane into nanodiscs (SMA-lipid particles, SMALPs), which contain membrane proteins possibly surrounded by their native lipid environment. We examined this approach for studies on several types of T cell membrane proteins, previously defined as raft or non-raft associated, to see whether the properties of the raft derived SMALPs differ from non-raft SMALPs. Our results indicate that two types of raft proteins, GPI-anchored proteins and two Src family kinases, are markedly present in membrane fragments much larger (>250?nm) than those containing non-raft proteins (<20?nm). Lipid probes sensitive to membrane fluidity (membrane order) indicate that the lipid environment in the large SMALPs is less fluid (more ordered) than in the small ones which may indicate the presence of a more ordered lipid L_o phase which is characteristic of membrane rafts. Also the lipid composition of the small vs. large SMALPs is markedly different – the large ones are enriched in cholesterol and lipids containing saturated fatty acids. In addition, we confirm that T cell membrane proteins present in SMALPs can be readily immunoisolated. Our results support the use of SMA as a potentially better (less artifact prone) alternative to detergents for studies on membrane proteins and their complexes, including membrane rafts.     

Lipid rafts are required for efficient signal transduction by CD1d

Plasma membranes of eukaryotic cells are not uniform, possessing distinct cholesterol- and sphingolipid-rich lipid raft microdomains which constitute critical sites for signal transduction through various immune cell receptors and their co-receptors. CD1d is a conserved family of major histocompatibility class I-like molecules, which has been established as an important factor in lipid antigen presentation to natural killer T (NKT) cells. Unlike conventional T cells, recognition of CD1d by the T cell receptor (TCR) of NKT cells does not require CD4 or CD8 co-receptors, which are critical for efficient TCR signaling. We found that murine CD1d (mCD1d) was constitutively present in the plasma membrane lipid rafts on antigen presenting cells, and that this restricted localization was critically important for efficient signal transduction to the target NKT cells, at low ligand densities, even without the involvement of co-receptors. Further our results indicate that there may be additional regulatory molecule(s), co-located in the lipid raft with mCD1d for NKT cell signaling.     

Ternary SNARE complexes are enriched in lipid rafts during mast cell exocytosis

Lipid rafts are membrane microdomains rich in cholesterol and glycosphingolipids that have been implicated in the regulation of intracellular protein trafficking. During exocytosis, a class of proteins termed SNAREs mediate secretory granule-plasma membrane fusion. To investigate the role of lipid rafts in secretory granule exocytosis, we examined the raft association of SNARE proteins and SNARE complexes in rat basophilic leukemia (RBL) mast cells. The SNARE protein SNAP-23 co-localized with a lipid raft marker and was present in detergent-insoluble lipid raft microdomains in RBL cells. By contrast, only small amounts (<20%) of the plasma membrane SNARE syntaxin 4 or the granule-associated SNARE vesicle-associated membrane protein (VAMP)-2 were present in these microdomains. Despite this, essentially all syntaxin 4 and most of VAMP-2 in these rafts were present in SNARE complexes containing SNAP-23, while essentially none of these complexes were present in nonraft membranes. Whereas SNAP-23 is membrane anchored by palmitoylation, the association of the transmembrane protein syntaxin 4 with lipid rafts was because of its binding to SNAP-23. After stimulating mast cells exocytosis, the amount of syntaxin 4 and VAMP-2 present in rafts increased twofold, and these proteins were now present in raft-associated phospho-SNAP-23/syntaxin 4/VAMP-2 complexes, revealing differential association of SNARE fusion complexes during the process of regulated exocytosis.     

Regulation of TrkB receptor translocation to lipid rafts by adenosine A2A receptors and its functional implications for BDNF-induced regulation of synaptic plasticity

Brain-derived neurotrophic factor (BDNF) signalling is critical for neuronal development and transmission. Recruitment of TrkB receptors to lipid rafts has been shown to be necessary for the activation of specific signalling pathways and modulation of neurotransmitter release by BDNF. Since TrkB receptors are known to be modulated by adenosine A_2A receptor activation, we hypothesized that activation of A_2A receptors could influence TrkB receptor localization among different membrane microdomains. We found that adenosine A_2A receptor agonists increased the levels of TrkB receptors in the lipid raft fraction of cortical membranes and potentiated BDNF-induced augmentation of phosphorylated TrkB levels in lipid rafts. Blockade of the clathrin-mediated endocytosis with monodansyl cadaverine (100 μM) did not modify the effects of the A_2A receptor agonists, but significantly impaired BDNF effects on TrkB recruitment to lipid rafts. The effect of A_2A receptor activation in TrkB localization was mimicked by 5 μM forskolin, an adenylyl cyclase activator. Also, it was blocked by the PKA inhibitors Rp-cAMPs and PKI-(14-22) and by the Src-family kinase inhibitor PP2. Moreover, removal of endogenous adenosine or disruption of lipid rafts reduced BDNF stimulatory effects on glutamate release from cortical synaptosomes. Lipid raft integrity was also required for the effects of BDNF upon hippocampal long-term potentiation at CA1 synapses. Our data demonstrate, for the first time, a BDNF-independent recruitment of TrkB receptors to lipid rafts, induced by the activation of adenosine A_2A receptors, with functional consequences for TrkB phosphorylation and BDNF-induced modulation of neurotransmitter release and hippocampal plasticity.     

Differently anchored influenza hemagglutinin mutants display distinct interaction dynamics with mutual rafts

Lipid rafts play important roles in cellular functions through concentrating or sequestering membrane proteins. This requires proteins to differ in the stability of their interactions with lipid rafts. However, knowledge of the dynamics of membrane protein-raft interactions is lacking. We employed FRAP to measure in live cells the lateral diffusion of influenza hemagglutinin (HA) proteins that differ in raft association. This approach can detect weak interactions with rafts not detectable by biochemical methods. Wild-type (wt) HA and glycosylphosphatidylinositol (GPI)-anchored HA (BHA-PI) diffused slower than a nonraft HA mutant, but became equal to the latter after cholesterol depletion. When antigenically distinct BHA-PI and wt HA were coexpressed, aggregation of BHA-PI into immobile patches reduced wt HA diffusion rate, suggesting transient interactions with BHA-PI raft patches. Conversely, patching wt HA reduced the mobile fraction of BHA-PI, indicating stable interactions with wt HA patches. Thus, the anchoring mode determines protein-raft interaction dynamics. GPI-anchored and transmembrane proteins can share the same rafts, and different proteins can interact stably or transiently with the same raft domains.     

Lipid rafts in T cell receptor signalling

The molecular events and the protein components that are involved in signalling by the T cell receptor (TCR) for antigen have been extensively studied. Activation of signalling cascades following TCR stimulation depends on the phosphorylation of the receptor by the tyrosine kinase Lck, which localizes to the cytoplasmic face of the plasma membrane by virtue of its post-translational modification. However, the precise order of events during TCR phosphorylation at the plasma membrane, remains to be defined. A current theory that describes early signalling events incorporates the function of lipid rafts, microdomains at the plasma membrane with distinct lipid and protein composition. Lipid rafts have been implicated in diverse biological functions in mammalian cells. In T cells, molecules with a key role in TCR signalling, including Lck, localize to these domains. Importantly, mutant versions of these proteins which fail to localise to raft domains were unable to support signalling by the TCR. Biochemical studies using purified detergent-resistant membranes (DRM) and confocal microscopy have suggested that upon stimulation, the TCR and Lck-containing lipid rafts may come into proximity allowing phosphorylation of the receptor. Further, there are data suggesting that phosphorylation of the TCR could depend on a transient increase in Lck activity that takes place within lipid rafts to initiate signalling. Current results and a model of how lipid rafts may regulate TCR signalling are discussed.