Spatiotemporal analysis of differential Akt regulation in plasma membrane microdomains

As a central kinase in the phosphatidylinositol 3-kinase pathway, Akt has been the subject of extensive research; yet, spatiotemporal regulation of Akt in different membrane microdomains remains largely unknown. To examine dynamic Akt activity in membrane microdomains in living cells, we developed a specific and sensitive fluorescence resonance energy transfer-based Akt activity reporter, AktAR, through systematic testing of different substrates and fluorescent proteins. Targeted AktAR reported higher Akt activity with faster activation kinetics within lipid rafts compared with nonraft regions of plasma membrane. Disruption of rafts attenuated platelet-derived growth factor (PDGF)-stimulated Akt activity in rafts without affecting that in nonraft regions. However, in insulin-like growth factor-1 (IGF)-1 stimulation, Akt signaling in nonraft regions is dependent on that in raft regions. As a result, cholesterol depletion diminishes Akt activity in both regions. Thus, Akt activities are differentially regulated in different membrane microdomains, and the overall activity of this oncogenic pathway is dependent on raft function. Given the increased abundance of lipid rafts in some cancer cells, the distinct Akt-activating characteristics of PDGF and IGF-1, in terms of both effectiveness and raft dependence, demonstrate the capabilities of different growth factor signaling pathways to transduce differential oncogenic signals across plasma membrane.     

Polyunsaturated eicosapentaenoic acid displaces proteins from membrane rafts by altering raft lipid composition

Polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (20:5 (n-3)) inhibit T lymphocyte activation probably by displacing acylated signaling proteins from membrane lipid rafts. Under physiological conditions, saturated fatty acyl residues of such proteins partition into the cytoplasmic membrane lipid leaflet with high affinity for rafts that are enriched in saturated fatty acyl-containing lipids. However, the biochemical alteration causing displacement of acylated proteins from rafts in PUFA-treated T cells is still under debate but could principally be attributed to altered protein acylation or changes in raft lipid composition. We show that treatment of Jurkat T cells with polyunsaturated eicosapentaenoic acid (20:5 (n-3)) results in marked enrichment of PUFAs (20:5; 22:5) in lipids from isolated rafts. Moreover, PUFAs were significantly incorporated into phosphatidylethanolamine that predominantly resides in the cytoplasmic membrane lipid leaflet. Notably, palmitate-labeled Src family kinase Lck and the linker for activation of T cells (LAT) were both displaced from lipid rafts indicating that acylation by PUFAs is not required for protein displacement from rafts in PUFA-treated T cells. In conclusion, these data provide strong evidence that displacement of acylated proteins from rafts in PUFA-treated T cells is predominantly due to altered raft lipid composition.     

The B cell-specific major raft protein,Raftlin, is necessary for the integrity of lipid raft and BCR signal transduction

Recent evidence indicates that membrane microdomains, termed lipid rafts, have a role in B-cell activation as platforms for B-cell antigen receptor (BCR) signal initiation. To gain an insight into the possible functioning of lipid rafts in B cells, we applied liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) methodologies to the identification of proteins that co-purified with lipid rafts of Raji cells. Among these raft proteins, we characterized a novel protein termed Raftlin (raft-linking protein). Like the Src family kinase, Raftlin is localized exclusively in lipid rafts by fatty acylation of N-terminal Gly2 and Cys3, and is co-localized with BCR before and after BCR stimulation. Disruption of the Raftlin gene in the DT40 B-cell line resulted in a marked reduction in the quantity of lipid raft components, including Lyn and ganglioside GM1, while overexpression of Raftlin increased the content of raft protein. Moreover, BCR-mediated tyrosine phosphorylation and calcium mobilization were impaired by the lack of Raftlin and actually potentiated by overexpression of Raftlin. These data suggest that Raftlin plays a pivotal role in the formation and/or maintenance of lipid rafts, therefore regulating BCR-mediated signaling.     

Two-dimensional electrophoretic analysis reveals that lipid rafts are intact at physiological temperature

Different proteins are found in lipid rafts depending on the isolation method. For example, insulin receptor was predominantly found in lipid raft fractions prepared from HepG2 cells with Brij 35, but were not present in lipid rafts isolated with Triton X-100. In order to assess the effect of detergent type and temperature on raft isolation, raft proteins from HepG2 cells were analyzed by two-dimensional (2-D) electrophoresis. More raft protein spots appeared when rafts were isolated by Brij 35 than by Triton X-100. In addition, more raft proteins were found when isolated at 37 degrees C than at 4 degrees C, indicating that lipid rafts are much more stable at physiological temperature (37 degrees C) in the presence of detergents. Indeed, lipid-modified proteins, such as Src and Lyn, were found in raft fractions even when detergent-resistant rafts were isolated at room or physiological temperature. The 2-D gel profile of raft proteins isolated with detergent-free (high-pH/carbonate) method was considerably similar to that of detergent-resistant raft proteins but contained a greater number of distinct protein spots. Whereas many detergent-resistant raft proteins disappeared upon cellular exposure to methyl-beta-cyclodextrin, high pH/carbonate-resistant raft proteins did not, suggesting that many of proteins isolated by high pH/carbonate could be contaminants. Considering these data, we conclude that liquid-ordered state of detergent-resistant lipid rafts is not destroyed at physiological temperature.     

Lipid rafts in health and disease

Lipid rafts are sphingolipid- and cholesterol-rich domains of the plasma membrane which contain a variety of signalling and transport proteins. Different subtypes of lipid rafts can be distinguished according to their protein and lipid composition. Caveolae are types of rafts that are rich in proteins of the caveolin family (caveolin-1, -2 and -3) which present a distinct signalling platform. The importance of lipid raft signalling in the pathogenesis of a variety of conditions, such as Alzheimer's, Parkinson's, cardiovascular and prion diseases, systemic lupus erythematosus and HIV, has been elucidated over recent years and makes these specific membrane domains an interesting target for pharmacological approaches in the cure and prevention of these diseases. This Review analyses the importance of lipid raft proteins and lipids in health and disease, with a focus on the current state of knowledge.     

Sphingolipid-cholesterol rafts diffuse as small entities in the plasma membrane of mammalian cells

To probe the dynamics and size of lipid rafts in the membrane of living cells, the local diffusion of single membrane proteins was measured. A laser trap was used to confine the motion of a bead bound to a raft protein to a small area (diam < or = 100 nm) and to measure its local diffusion by high resolution single particle tracking. Using protein constructs with identical ectodomains and different membrane regions and vice versa, we demonstrate that this method provides the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI) -anchored and transmembrane proteins are raft-associated, their diffusion becomes independent of the type of membrane anchor and is significantly reduced compared with that of nonraft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins for transmembrane raft proteins to the level of transmembrane nonraft proteins and for GPI-anchored proteins even further. Raft-associated GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 min. The measurements agree with lipid rafts being cholesterol-stabilized complexes of 26 +/- 13 nm in size diffusing as one entity for minutes.     

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.     

Global Network Analysis of Lipid-Raft-Related Proteins Reveals Their Centrality in the Network and Their Roles in Multiple Biological Processes

Lipid rafts are specialized cholesterol-enriched microdomains in the cell membrane. They have been known as a platform for protein-protein interactions and to take part in multiple biological processes. Nevertheless, how lipid rafts influence protein properties at the proteomic level is still an open question for researchers using traditional biochemical approaches. Here, by annotating the lipid raft localization of proteins in human protein-protein interaction networks, we performed a systematic analysis of the function of proteins related to lipid rafts. Our results demonstrated that lipid raft proteins and their interactions were critical for the structure and stability of the whole network, and that the interactions between them were significantly enriched. Furthermore, for each protein in the network, we calculated its “lipid raft dependency (LRD),” which indicates how close it is topologically associated with lipid rafts, and we then uncovered the connection between LRD and protein functions. Proteins with high LRD tended to be essential for mammalian development, and malfunction of these proteins was inclined to cause human diseases. Coordinated with their neighbors, high-LRD proteins participated in multiple biological processes and targeted many pathways in diseases pathogenesis. High-LRD proteins were also found to have tissue specificity of expression. In summary, our network-based analysis denotes that lipid raft proteins have higher centrality in the network, and that lipid-raft-related proteins have multiple functions and are probably concerned with many biological processes in disease development.     

Regulation of integrin growth factor interactions in oligodendrocytes by lipid raft microdomains

Individual growth factors can regulate multiple aspects of behavior within a single cell during differentiation, with each signaling pathway controlled independently and also responsive to other receptors such as cell surface integrins. The mechanisms by which this is achieved remain poorly understood. Here we use myelin-forming oligodendrocytes and their precursors to examine the role of lipid rafts, cholesterol and sphingolipid-rich microdomains of the cell membrane implicated in cell signaling. In these cells, the growth factor PDGF has sequential and independent roles in proliferation and survival. We show that the oligodendrocyte PDGFalpha receptor becomes sequestered in a raft compartment at the developmental stage when PDGF ceases to promote proliferation, but is now required for survival. We also show that laminin-2, which is expressed on axons in the CNS and which provides a target-dependent signal for oligodendrocyte survival by amplification of PDGFalphaR signaling, induces clustering of the laminin binding integrin alpha6beta1 with the PDGFalphaR-containing lipid raft domains. This extracellular matrix-induced colocalization of integrin and growth factor receptor generates a signaling environment within the raft for survival-promoting PI3K/Akt activity. These results demonstrate novel signaling roles for lipid rafts that ensure the separation and amplification of growth factor signaling pathways during development.     

Lipid raft proteomics: analysis of in-solution digest of sodium dodecyl sulfate-solubilized lipid raft proteins by liquid chromatography-matrix-assisted laser desorption/ionization tandem mass spectrometry

Lipid rafts are glycolipid- and cholesterol-enriched membrane microdomains implicated in membrane signaling and trafficking. The highly hydrophobic nature of lipid raft proteins pose significant problems of solubilization and recovery that hinder analysis by mass spectrometry (MS) and may under-report the composition of lipid rafts. In a previous investigation of the monocyte lipid raft in which proteins were digested with trypsin following polyacrylamide gel electrophoresis we identified 52 proteins. Here we report the development of a sodium dodecyl sulfate (SDS)-aided approach in which proteins are digested in solution and examined by high-performance liquid chromatography-matrix-assisted laser desorption/ionization-tandem mass spectrometry (HPLC-MALDI-MS/MS) using a novel LC-MALDI interface thereby circumventing the need to separate proteins on gels. Using this approach we identified 71 proteins in the lipid raft, 45 of which were not detected using in-gel digestion. Among the new proteins are alpha- and beta-tubulin, tubulinspecific chaperone A, a folding protein involved in tubulin dimer assembly, and KIF13, a microtubule motor protein indicating that proteins involved in microtubule assembly and trafficking are more readily detected using an in-solution approach. To investigate why tubulin was not identified by in-gel digestion, we compared the distribution of alpha-tubulin and the raft marker flotillin-2 in buoyant density gradients before and after separation on SDS-gels. Both proteins were present in the raft fractions, but tubulin was selectively lost following separation on SDS-gels. Assemblies of cytoskeletal proteins with lipid rafts may therefore be resolved using in-solution digestion that would be missed using gel-based approaches.     

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.     

Cholesterol efflux alters lipid raft stability and distribution during capacitation of boar spermatozoa

A reduction in plasma membrane cholesterol is one of the early events that either triggers or is closely associated with capacitation of mammalian spermatozoa. In this investigation, we have examined the effects of cholesterol efflux on tyrosine phosphorylation, lipid diffusion, and raft organization in boar spermatozoa. Results show that a low level of cholesterol efflux, mediated by 5 mM methyl-beta-cyclodextrin (MBCD), enhances capacitation and induces phosphorylation of two proteins at 26 and 15 kDa without affecting sperm viability. Lipid diffusion rates under these conditions are largely unaffected except when cholesterol efflux is excessive. Low-density Triton X100-insoluble complexes (lipid rafts) were isolated from spermatozoa and found to have a restricted profile of proteins. Capacitation-associated cholesterol efflux has no effect on raft composition, but cholesterol depletion destabilizes them completely and phosphorylation is suppressed. During MBCD-mediated capacitation, the distribution of GM1 gangliosides on spermatozoa changes in a sequential manner from overlying the sperm tail to clustering on the sperm head. It is concluded that there is a safe window for removal of plasma membrane cholesterol from spermatozoa within which protein phosphorylation and polarized migration of lipid rafts take place. A preferential loss of cholesterol from the nonraft pool may be the stimulus that promotes raft clustering over the anterior sperm head.     

A lipid raft environment enhances Lyn kinase activity by protecting the active site tyrosine from dephosphorylation

The plasma membrane contains ordered lipid domains, commonly called lipid rafts, enriched in cholesterol, sphingolipids, and certain signaling proteins. Lipid rafts play a structural role in signal initiation by the high affinity receptor for IgE. Cross-linking of IgE-receptor complexes by antigen causes their coalescence with lipid rafts, where they are phosphorylated by the Src family tyrosine kinase, Lyn. To understand how lipid rafts participate in functional coupling between Lyn and FcepsilonRI, we investigated whether the lipid raft environment influences the specific activity of Lyn. We used differential detergent solubility and sucrose gradient fractionation to isolate Lyn from raft and nonraft regions of the plasma membrane in the presence or absence of tyrosine phosphatase inhibitors. We show that Lyn recovered from lipid rafts has a substantially higher specific activity than Lyn from nonraft environments. Furthermore, this higher specific activity correlates with increased tyrosine phosphorylation at the active site loop of the kinase domain. Based on these results, we propose that lipid rafts exclude a phosphatase that negatively regulates Lyn kinase activity by constitutive dephosphorylation of the kinase domain tyrosine residue of Lyn. In this model, cross-linking of FcepsilonRI promotes its proximity to active Lyn in a lipid raft environment.     

Phosphotyrosine protein dynamics in cell membrane rafts of sphingosine-1-phosphate-stimulated human endothelium: Role in barrier enhancement

Sphingosine-1-phosphate (S1P), a lipid growth factor, is critical to the maintenance and enhancement of vascular barrier function via processes highly dependent upon cell membrane raft-mediated signaling events. Anti-phosphotyrosine 2 dimensional gel electrophoresis (2-DE) immunoblots confirmed that disruption of membrane raft formation (via methyl-β-cyclodextrin) inhibits S1P-induced protein tyrosine phosphorylation. To explore S1P-induced dynamic changes in membrane rafts, we used 2-D techniques to define proteins within detergent-resistant cell membrane rafts which are differentially expressed in S1P-challenged (1 μM, 5 min) human pulmonary artery endothelial cells (EC), with 57 protein spots exhibiting > 3-fold change. S1P induced the recruitment of over 20 cell membrane raft proteins exhibiting increasing levels of tyrosine phosphorylation including known barrier-regulatory proteins such as focal adhesion kinase (FAK), cortactin, p85α phosphatidylinositol 3-kinase (p85αPI3K), myosin light chain kinase (nmMLCK), filamin A/C, and the non-receptor tyrosine kinase, c-Abl. Reduced expression of either FAK, MLCK, cortactin, filamin A or filamin C by siRNA transfection significantly attenuated S1P-induced EC barrier enhancement. Furthermore, S1P induced cell membrane raft components, p-caveolin-1 and glycosphingolipid (GM1), to the plasma membrane and enhanced co-localization of membrane rafts with p-caveolin-1 and p-nmMLCK. These results suggest that S1P induces both the tyrosine phosphorylation and recruitment of key actin cytoskeletal proteins to membrane rafts, resulting in enhanced human EC barrier function.     

Oxidation-reduction respiratory chains and ATP synthase complex are localized in detergent-resistant lipid rafts

In order to detect and identify ubiquitous lipid raft marker proteins, we isolated lipid rafts from different mouse organs, including the liver, lung, large brain, and kidney, and analyzed their proteins via 2-DE. Many protein spots were determined to be ubiquitous in all of the lipid rafts, and were annotated via LC and MS/MS. Twelve proteins were identified as ubiquitous raft proteins, and most of these were determined to be mitochondrial proteins, including mortalin, prohibitin, voltage-dependent anion channel, ATP synthase, NADH dehydrogenase, and ubiquinol-cytochrome c reductase. Via immunoblotting, these proteins were shown to exist in detergent-resistant lipid rafts prepared using different organ tissues. Since these oxidation-reduction respiratory chains and ATP synthase complex were detected in detergent-resistant lipid raft fractions which had been isolated from the plasma membrane but not from the mitochondria, and found in the cell surface when determined by immunofluoresence and immunohistochemistry, we conclude that plasma membrane lipid rafts might contain oxidation-reduction respiratory chains and ATP synthase complex.     

Platelet-derived growth factor and heparin-like glycosaminoglycans regulate thrombospondin synthesis and deposition in the matrix by smooth muscle cells

Platelet-derived growth factor (PDGF), a smooth muscle cell (SMC) mitogen, and heparin-like glycosaminoglycans, known inhibitors of SMC growth and migration, were found to regulate thrombospondin synthesis and matrix deposition by cultured rat aortic SMC. The synthesis and distribution of thrombospondin was examined in growth-arrested SMCs, in PDGF-stimulated SMCs, and in heparin-treated SMCs using metabolic labeling and immunofluorescence techniques. Thrombospondin synthesis in response to purified PDGF occurred within 1 h after addition of growth factor to growth-arrested SMCs, peaked at 2 h, and returned to baseline levels by 5 h. The induction of synthesis of thrombospondin by PDGF was dose dependent, with a maximal effect observed at 2.5 ng/ml. Actinomycin D (2 micrograms/ml) inhibited thrombospondin induction by PDGF, suggesting a requirement for new RNA synthesis. In the presence of heparin and related polyanions, the incorporation of thrombospondin into the SMC extracellular matrix was markedly reduced. This effect was dose dependent with a maximal effect observed at a heparin concentration of 1 microgram/ml. Heparin did not affect the ability of SMCs to synthesize thrombospondin in response to PDGF. We interpret these data to suggest a role for thrombospondin in the SMC proliferative response to PDGF and in the regulation of SMC growth and migration by glycosaminoglycans.     

The low-density lipoprotein receptor-related protein-1 associates transiently with lipid rafts

The low-density lipoprotein receptor-related protein-1 (LRP-1) is a multifunctional receptor that undergoes constitutive endocytosis and recycling. To identify LRP-1 in lipid rafts, we biotin-labeled cells using a membrane-impermeable reagent and prepared Triton X-100 fractions. Raft-associated proteins were identified in streptavidin affinity-precipitates of the Triton X-100-insoluble fraction. PDGF beta-receptor was identified exclusively in lipid rafts, whereas transferrin receptor was excluded. LRP-1 distributed partially into rafts in murine embryonic fibroblasts (MEFs) and HT 1080 cells, but not in smooth muscle cells and CHO cells. LRP-1 partitioning into rafts was not altered by ligands, including alpha2-macroglobulin, platelet-derived growth factor-BB, and receptor-associated protein (RAP). To examine LRP-1 trafficking between membrane microdomains, we developed a novel method based on biotinylation and detergent fractionation. Association of LRP-1 with rafts was transient; by 15 min, nearly all of the LRP-1 that was initially raft-associated exited this compartment. LRP-1 in the Triton X-100-soluble fraction, which excludes lipid rafts, demonstrated complex kinetics, with phases reflecting import from rafts, endocytosis, and recycling. Potassium depletion blocked LRP-1 endocytosis but did not inhibit trafficking of LRP-1 from rafts into detergent-soluble microdomains. Our data support a model in which LRP-1 transiently associates with rafts but does not form a stable pool. Fluid movement of LRP-1 between microdomains may facilitate its function in promoting the endocytosis of other plasma membrane proteins, such as the urokinase receptor, which localizes in lipid rafts.     

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.     

Increased phosphorylation and redistribution of NMDA receptors between synaptic lipid rafts and post-synaptic densities following transient global ischemia in the rat brain

Ischemia results in increased phosphorylation of NMDA receptors. To investigate the possible role of lipid rafts in this increase, lipid rafts and post-synaptic densities (PSDs) were isolated by the extraction of rat brain synaptosomes with Triton X-100 followed by sucrose density gradient centrifugation. Lipid rafts accounted for the majority of PSD-95, whereas SAP102 was predominantly located in PSDs. Between 50 and 60% of NMDA receptors were associated with lipid rafts. Greater than 85-90% of Src and Fyn were present in lipid rafts, whereas Pyk2 was mainly associated with PSDs. Lipid rafts and PSDs were isolated from animals subjected to 15 min of global ischemia followed by 6 h of recovery. Ischemia did not affect the yield, density, flotillin-1 or cholesterol content of lipid rafts. Following ischemia, the phosphorylation of NR1 by protein kinase C and tyrosine phosphorylation of NR2A and NR2B was increased in both lipid rafts and PSDs, with a greater increase in tyrosine phosphorylation occurring in the raft fraction. Following ischemia, NR1, NR2A and NR2B levels were elevated in PSDs and reduced in lipid rafts. The findings are consistent with a model involving close interaction between lipid rafts and PSDs and a role for lipid rafts in ischemia-induced signaling pathways.     

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.