Targeting of voltage-gated calcium channel α2δ-1 subunit to lipid rafts is independent from a GPI-anchoring motif

Voltage-gated calcium channels (Ca(v)) exist as heteromultimers comprising a pore-forming α(1) with accessory β and α(2)δ subunits which modify channel trafficking and function. We previously showed that α(2)δ-1 (and likely the other mammalian α(2)δ isoforms--α(2)δ-2, 3 and 4) is required for targeting Ca(v)s to lipid rafts, although the mechanism remains unclear. Whilst originally understood to have a classical type I transmembrane (TM) topology, recent evidence suggests the α(2)δ subunit contains a glycosylphosphatidylinositol (GPI)-anchor that mediates its association with lipid rafts. To test this notion, we have used a strategy based on the expression of chimera, where the reported GPI-anchoring sequences in the gabapentinoid-sensitive α(2)δ-1 subunit have been substituted with those of a functionally inert Type I TM-spanning protein--PIN-G. Using imaging, electrophysiology and biochemistry, we find that lipid raft association of PIN-α(2)δ is unaffected by substitution of the GPI motif with the TM domain of PIN-G. Moreover, the presence of the GPI motif alone is not sufficient for raft localisation, suggesting that upstream residues are required. GPI-anchoring is susceptible to phosphatidylinositol-phospholipase C (PI-PLC) cleavage. However, whilst raft localisation of PIN-α(2)δ is disrupted by PI-PLC treatment, this is assay-dependent and non-specific effects of PI-PLC are observed on the distribution of the endogenous raft marker, caveolin, but not flotillin. Taken together, these data are most consistent with a model where α(2)δ-1 retains its type I transmembrane topology and its targeting to lipid rafts is governed by sequences upstream of the putative GPI anchor, that promote protein-protein, rather than lipid-lipid interactions.     

Identification of a disulfide bridge essential for structure and function of the voltage-gated Ca(2+) channel α(2)δ-1 auxiliary subunit

Voltage-gated calcium (Ca(V)) channels are transmembrane proteins that form Ca(2+)-selective pores gated by depolarization and are essential regulators of the intracellular Ca(2+) concentration. By providing a pathway for rapid Ca(2+) influx, Ca(V) channels couple membrane depolarization to a wide array of cellular responses including neurotransmission, muscle contraction and gene expression. Ca(V) channels fall into two major classes, low voltage-activated (LVA) and high voltage-activated (HVA). The ion-conducting pathway of HVA channels is the α(1) subunit, which typically contains associated β and α(2)δ ancillary subunits that regulate the properties of the channel. Although it is widely acknowledged that α(2)δ-1 is post-translationally cleaved into an extracellular α(2) polypeptide and a membrane-anchored δ protein that remain covalently linked by disulfide bonds, to date the contribution of different cysteine (Cys) residues to the formation of disulfide bridges between these proteins has not been investigated. In the present report, by predicting disulfide connectivity with bioinformatics, molecular modeling and protein biochemistry experiments we have identified two Cys residues involved in the formation of an intermolecular disulfide bond of critical importance for the structure and function of the α(2)δ-1 subunit. Site directed-mutagenesis of Cys404 (located in the von Willebrand factor-A region of α(2)) and Cys1047 (in the extracellular domain of δ) prevented the association of the α(2) and δ peptides upon proteolysis, suggesting that the mature protein is linked by a single intermolecular disulfide bridge. Furthermore, co-expression of mutant forms of α(2)δ-1 Cys404Ser and Cys1047Ser with recombinant neuronal N-type (Ca(V)2.2α(1)/β(3)) channels, showed decreased whole-cell patch-clamp currents indicating that the disulfide bond between these residues is required for α(2)δ-1 function.     

Involvement of lipid rafts in macrophage apoptosis induced by cationic liposomes

We have demonstrated that protein kinase Cδ (PKCδ) could be involved in macrophage apoptosis induced by cationic liposomes composed of stearylamine (SA-liposomes), but the detailed mechanism of how SA-liposomes activate PKCδ has remained unclear. In this paper, we clarified whether lipid rafts are involved in the PKCδ activation induced by SA-liposomes. Co-localization of SA-liposomes and Cholera toxin B subunit (CBT), which specifically binds to ganglioside GM1 on lipid rafts, was found by microscopic observation. The incorporation of SA-liposomes into lipid rafts was clearly inhibited by the pretreatment of cells with an agent, 2,6-di-O-methyl-α-cyclodextrin (DM-α-CD) which disrupts lipid rafts. Activation of PKCδ and externalization of phosphatidylserine induced by SA-liposomes were also suppressed by DM-α-CD, which extracts sphingolipids and proteins from lipid rafts. Reactive oxygen species (ROS) generation, which could be involved in the macrophage apoptosis, was also inhibited by DM-α-CD. Furthermore, apoptosis induced by SA-liposomes was clearly inhibited when the cells were pre-treated with DM-α-CD, but not nystatin, a cholesterol-sequestering agent that disrupt lipid rafts. These findings suggest that sphingolipids in lipid rafts are involved in the activation of PKCδ which leads to apoptosis induced by cationic liposomes, SA-liposomes.     

Regulation of SGLT expression and localization through Epac/PKA-dependent caveolin-1 and F-actin activation in renal proximal tubule cells

This study demonstrated that exchange proteins directly activated by cAMP (Epac) and protein kinase A (PKA) by 8-bromo (8-Br)-adenosine 3',5'-cyclic monophosphate (cAMP) stimulated [(14)C]-α-methyl-D-glucopyranoside (α-MG) uptake through increased sodium-glucose cotransporters (SGLTs) expression and translocation to lipid rafts in renal proximal tubule cells (PTCs). In PTCs, SGLTs were colocalized with lipid raft caveolin-1 (cav-1), disrupted by methyl-β-cyclodextrin (MβCD). Selective activators of Epac or PKA, 8-Br-cAMP, and forskolin stimulated expressions of SGLTs and α-MG uptake in PTCs. In addition, 8-Br-cAMP-induced PKA and Epac activation increased phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and nuclear factor kappa B (NF-κB), which were involved in expressions of SGLTs. Furthermore, 8-Br-cAMP stimulated SGLTs translocation to lipid rafts via filamentous actin (F-actin) organization, which was blocked by cytochalasin D. In addition, cav-1 and SGLTs stimulated by 8-Br-cAMP were detected in lipid rafts, which were blocked by cytochalasin D. Furthermore, 8-Br-cAMP-induced SGLTs translocation and α-MG uptake were attenuated by inhibition of cav-1 activation with cav-1 small interfering RNA (siRNA) and inhibition of F-actin organization with TRIO and F-actin binding protein (TRIOBP). In conclusion, 8-Br-cAMP stimulated α-MG uptake via Epac and PKA-dependent SGLTs expression and trafficking through cav-1 and F-actin in PTCs.     

Time course and specificity of the pharmacological disruption of the trafficking of voltage-gated calcium channels by gabapentin

The mechanism of action of gabapentin is still not well understood. It binds to the α2δ-1 and α2δ-2 subunits of voltage-gated calcium channels but has little acute effect on calcium currents in several systems. However, our recent results conclusively demonstrated that gabapentin inhibited calcium currents when applied chronically but not acutely, both in heterologous expression systems and in dorsal root ganglion neurons 1. In that study we only examined a 40 hour time point of incubation with gabapentin, and here we have extended these results to include the effect of up to 6 and 20 hours incubation with gabapentin on calcium channel currents formed from CaV2.1/β4/α2δ-2 subunits. Gabapentin was significantly effective to inhibit the currents if included for 17-20 hours prior to recording, but it did not produce a significant inhibition if included for 3-6 hours. We previously concluded that gabapentin acts primarily at an intracellular location, requiring uptake into cells. However, this effect is mediated by α2δ subunits, being prevented by mutations in either α2δ-1 or α2δ-2 that abolish gabapentin binding 1. Furthermore, we also showed that the trafficking of α2δ-2 and CaV2 channels was disrupted by gabapentin. Here we have also extended that study, to show that the cell-surface expression of CaV2.1 is not reduced by chronic gabapentin if it is co-expressed with α2δ-2 containing a point mutation (R282A) that prevents gabapentin binding     

Potency of GABA at human recombinant GABAA receptors expressed in Xenopus oocytes: a mini review

GABA_A receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory neurotransmission in the central nervous system. They are thought to be composed of 2 alpha (α), 2 beta (β) subunits and one other such as a gamma (γ) or delta (δ) subunit. The potency of GABA is influenced by the subunit composition. However, there are no reported systematic studies that evaluate GABA potency on a comprehensive number of subunit combinations expressed in Xenopus oocytes, despite the wide use of this heterologous expression system in structure–function studies and drug discovery. Thus, the aim of this study was to conduct a systematic characterization of the potency of GABA at 43 human recombinant GABA_A receptor combinations expressed in Xenopus oocytes using the two-electrode voltage clamp technique. The results show that the α-subunits and to a lesser extent, the β-subunits influence GABA potency. Of the binary and ternary combinations with and without the γ2L subunit, the α6/γ2L-containing receptors were the most sensitive to GABA, while the β2- or β3-subunit conferred higher sensitivity to GABA than receptors containing the β1-subunit with the exception of the α2β1γ2L and α6β1γ2L subtypes. Of the δ-subunit containing GABA_A receptors, α4/δ-containing GABA_A receptors displayed highest GABA sensitivity, with mid-nanomolar concentrations activating α4β1δ and α4β3δ receptors. At α4β2δ, GABA had low micromolar activity.     

αVβ3-integrin relocalizes nectin1 and routes herpes simplex virus to lipid rafts

Herpes simplex virus (HSV) enters cells by fusion at plasma membranes or endosomes. Cellular factors route the virus to different pathways. αVβ3-integrin directs HSV to a lipid raft and acidic endosome pathway. We report that infection mediated by nectin1 plus αVβ3-integrin exhibits the same characteristics as entry mediated by raft-located forms of nectin. αVβ3-integrin relocalizes nectin1 to lipid rafts, independently of virus. Thus, HSV routing to the lipid raft-dependent pathway is consequent to the integrin-induced relocalization of nectin1. Inhibition by the Na+/H+ exchanger 5-(N-ethyl-N-isopropyl)amirolide suggests that αVβ3-integrin overexpression favors HSV macropinocytic uptake in some cells but not in others.     

Activation of myeloid cell-specific adhesion class G protein-coupled receptor EMR2 via ligation-induced translocation and interaction of receptor subunits in lipid raft microdomains

The adhesion class G protein-coupled receptors (adhesion-GPCRs) play important roles in diverse biological processes ranging from immunoregulation to tissue polarity, angiogenesis, and brain development. These receptors are uniquely modified by self-catalytic cleavage at a highly conserved GPCR proteolysis site (GPS) dissecting the receptor into an extracellular subunit (α) and a seven-pass transmembrane subunit (β) with cellular adhesion and signaling functions, respectively. Using the myeloid cell-restricted EMR2 receptor as a paradigm, we exam the mechanistic relevance of the subunit interaction and demonstrate a critical role for GPS autoproteolysis in mediating receptor signaling and cell activation. Interestingly, two distinct receptor complexes are identified as a result of GPS proteolysis: one consisting of a noncovalent α-β heterodimer and the other comprising two completely independent receptor subunits which distribute differentially in membrane raft microdomains. Finally, we show that receptor ligation induces subunit translocation and colocalization within lipid rafts, leading to receptor signaling and inflammatory cytokine production by macrophages. Our present data resolve earlier conflicting results and provide a new mechanism of receptor signaling, as well as providing a paradigm for signal transduction within the adhesion-GPCR family.     

Ba2+ currents in inner and outer hair cells of mice lacking the voltage-dependent Ca2+ channel subunits β3 or β4

Voltage-activated Ca2+ channels comprise complexes of a pore-forming Cavα1 and auxiliary subunits Cavβ, Cavα2δ and sometimes Cavγ. The intracellular Cavβ subunit assists in trafficking and surface expression of the Cavα1 subunit and can modulate biophysical properties of the Ca2+ channel. Four genes, Cavβ1-4, exist which confer different properties to Ca2+ currents through the various Cavα1 subunits. Ca2+ currents in cochlear inner (IHC) and outer hair cells (OHC) serving synaptic transmission flow predominantly through the L type Cavα1 subunit Cav1.3, but associated Cavβ subunits are unknown. In the organ of Corti, we found mRNA and protein for all four Cavβ subunits including Cavβ2, but clear assignment of the Cavβ1 4 immunolabelling with hair cells or nerve fibers was difficult. We analyzed Cavβ3 knockout (Cavβ3 / ) and Cavβ4 mutant mice (Cavβ4lh/lh), which had normal hearing. Recording voltage-activated Ba2+ currents from hair cells of the two mouse models revealed distinct significant changes of cell size and Ba2+ current properties compared with their wildtype controls. Neonatal Cavβ4lh/lh IHCs showed reduced membrane capacitances and changes in the voltage dependence and kinetics of current activation, whereas mature IHCs had reduced peak currents compared with Cavβ4wt, altogether indicating the presence of Cavβ4 in IHCs. Ba2+ currents of Cavβ3 / OHCs showed largely reduced amplitudes, changes in the voltage dependence and kinetics of Ba2+ current activation, and increased inactivation compared with Cavβ3wt, pointing to a role of Cavβ3 for OHCs. These results indicate that neither Cavβ3 nor Cavβ4 are indispensable for hair cell Ca2+ currents but contribute to the overall current properties.     

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.     

Single-channel monitoring of reversible L-type Ca(2+) channel Ca(V)α(1)-Ca(V)β subunit interaction

Voltage-dependent Ca(2+) channels are heteromultimers of Ca(V)α(1) (pore), Ca(V)β- and Ca(V)α(2)δ-subunits. The stoichiometry of this complex, and whether it is dynamically regulated in intact cells, remains controversial. Fortunately, Ca(V)β-isoforms affect gating differentially, and we chose two extremes (Ca(V)β(1a) and Ca(V)β(2b)) regarding single-channel open probability to address this question. HEK293α(1C) cells expressing the Ca(V)1.2 subunit were transiently transfected with Ca(V)α(2)δ1 alone or with Ca(V)β(1a), Ca(V)β(2b), or (2:1 or 1:1 plasmid ratio) combinations. Both Ca(V)β-subunits increased whole-cell current and shifted the voltage dependence of activation and inactivation to hyperpolarization. Time-dependent inactivation was accelerated by Ca(V)β(1a)-subunits but not by Ca(V)β(2b)-subunits. Mixtures induced intermediate phenotypes. Single channels sometimes switched between periods of low and high open probability. To validate such slow gating behavior, data were segmented in clusters of statistically similar open probability. With Ca(V)β(1a)-subunits alone, channels mostly stayed in clusters (or regimes of alike clusters) of low open probability. Increasing Ca(V)β(2b)-subunits (co-)expressed (1:2, 1:1 ratio or alone) progressively enhanced the frequency and total duration of high open probability clusters and regimes. Our analysis was validated by the inactivation behavior of segmented ensemble averages. Hence, a phenotype consistent with mutually exclusive and dynamically competing binding of different Ca(V)β-subunits is demonstrated in intact cells.     

Unexpected Properties of δ-Containing GABAA Receptors in Response to Ligands Interacting with the α+ β− Site

GABA_A receptors are the major inhibitory neurotransmitter receptors in the central nervous system and are the targets of many clinically important drugs, which modulate GABA induced chloride flux by interacting with separate and distinct allosteric binding sites. Recently, we described an allosteric modulation occurring upon binding of pyrazoloquinolinones to a novel binding site at the extracellular α+ β? interface. Here, we investigated the effect of 4-(8-methoxy-3-oxo-3,5-dihydro-2H-pyrazolo[4,3-c]quinolin-2-yl)benzonitrile (the pyrazoloquinolinone LAU 177) at several αβ, αβγ and αβδ receptor subtypes. LAU 177 enhanced GABA-induced currents at all receptors investigated, and the extent of modulation depended on the type of α and β subunits present within the receptors. Whereas the presence of a γ2 subunit within αβγ2 receptors did not dramatically change LAU 177 induced modulation of GABA currents compared to αβ receptors, we observed an unexpected threefold increase in modulatory efficacy of this compound at α1β2,3δ receptors. Steric hindrance experiments as well as inhibition by the functional α+ β? site antagonist LAU 157 indicated that the effects of LAU 177 at all receptors investigated were mediated via the α+ β? interface. The stronger enhancement of GABA-induced currents by LAU 177 at α1β3δ receptors was not observed at α4,6β3δ receptors. Other experiments indicated that this enhancement of modulatory efficacy at α1β3δ receptors was not observed with another α+ β? modulator, and that the efficacy of modulation by α+ β? ligands is influenced by all subunits present in the receptor complex and by structural details of the respective ligand.     

Influence of membrane components in the binding of proteins to membrane surfaces

We have quantified the enhancement of membrane binding of activated and deactivated Galpha(s) and Galpha(q) subunits, Gbetagamma subunits, and phospholipase Cbeta(2) by lipid rafts and by the presence of membrane-associated protein partners. Membrane binding studies show that lipid rafts do not affect the intrinsic membrane affinity of Galpha(q)(GDP) and Galpha(s)(GDP), supporting the idea that these proteins partition evenly between the domains. Visualization of lipid rafts on monolayers by use of a probe that does not enter raft domains shows that neither activated nor deactivated Galpha(q)(GDP) subunits distribute evenly between the raft and nonraft domains, contrary to previous suggestions. Membrane binding of deactivated Galpha(q) and Galpha(s)(GDP) became weaker when Gbetagamma subunits were present, in contrast with the behavior predicted by thermodynamics. However, activated Galpha subunits and phospholipase Cbeta(2) were recruited to membrane surfaces by protein partners by predicted amounts. Our studies suggest that the anomalous behavior seen for deactivated Galpha subunits in the presence of Gbetagamma subunits may be due to conformational changes in the N-terminus and/or occlusion of a portion of its membrane interaction region by Gbetagamma. Even though membrane recruitment was clearly observed for one protein partner, the presence of a second partner of lower affinity did not further promote membrane binding. For these proteins, the formation of larger protein complexes with very high membrane affinities is unlikely.     

Lipid rafts function in Ca2+ signaling responsible for activation of sperm motility and chemotaxis in the ascidian Ciona intestinalis

Lipid rafts are specialized membrane microdomains that function as signaling platforms across plasma membranes of many animal and plant cells. Although there are several studies implicating the role of lipid rafts in capacitation of mammalian sperm, the function of these structures in sperm motility activation and chemotaxis remains unknown. In the ascidian Ciona intestinalis, egg-derived sperm activating- and attracting-factor (SAAF) induces both activation of sperm motility and sperm chemotaxis to the egg. Here we found that a lipid raft disrupter, methyl-β-cyclodextrin (MCD), inhibited both SAAF-induced sperm motility activation and chemotaxis. MCD inhibited both SAAF-promoted synthesis of intracellular cyclic AMP and sperm motility induced by ionophore-mediated Ca(2+) entry, but not that induced by valinomycin-mediated hyperpolarization. Ca(2+)-imaging revealed that lipid raft disruption inhibited Ca(2+) influx upon activation of sperm motility. The Ca(2+)-activated adenylyl cyclase was clearly inhibited by MCD in isolated lipid rafts. The results suggest that sperm lipid rafts function in signaling upstream of cAMP synthesis, most likely in SAAF-induced Ca(2+) influx, and are required for Ca(2+)-dependent pathways underlying activation and chemotaxis in Ciona sperm.     

Cytoskeleton-mediated death receptor and ligand concentration in lipid rafts forms apoptosis-promoting clusters in cancer chemotherapy

While investigating the mechanism of action of the novel antitumor drug Aplidin, we have discovered a potent and novel cell-killing mechanism that involves the formation of Fas/CD95-driven scaffolds in membrane raft clusters housing death receptors and apoptosis-related molecules. Fas, tumor necrosis factor-receptor 1, and tumor necrosis factor-related apoptosis-inducing ligand receptor 2/death receptor 5 were clustered into lipid rafts in leukemic Jurkat cells following Aplidin treatment, the presence of Fas being essential for apoptosis. Preformed membrane-bound Fas ligand (FasL) as well as downstream signaling molecules, including Fas-associated death domain-containing protein, procaspase-8, procaspase-10, c-Jun amino-terminal kinase, and Bid, were also translocated into lipid rafts, connecting death receptor extrinsic and mitochondrial intrinsic apoptotic pathways. Blocking Fas/FasL interaction partially inhibited Aplidin-induced apoptosis. Aplidin was rapidly incorporated into membrane rafts, and drug uptake was inhibited by lipid raft disruption. Actin-linking proteins ezrin, moesin, RhoA, and RhoGDI were conveyed into Fas-enriched rafts in drug-treated leukemic cells. Disruption of lipid rafts and interference with actin cytoskeleton prevented Fas clustering and apoptosis. Thus, Aplidin-induced apoptosis involves Fas activation in both a FasL-independent way and, following Fas/FasL interaction, an autocrine way through the concentration of Fas, membrane-bound FasL, and signaling molecules in membrane rafts. These data indicate a major role of actin cytoskeleton in the formation of Fas caps and highlight the crucial role of the clusters of apoptotic signaling molecule-enriched rafts in apoptosis, acting as concentrators of death receptors and downstream signaling molecules and as the linchpin from which a potent death signal is launched.     

Lipid raft localization of cell surface E-selectin is required for ligation-induced activation of phospholipase C gamma

E-selectin, an endothelial cell surface adhesion receptor for leukocytes, also acts as a signaling receptor. Upon multivalent ligation, E-selectin transduces outside-in signals into the endothelium leading to changes in intracellular Ca(2+) concentration and activation of the mitogen-activated protein kinase signaling pathway. In addition, following leukocyte engagement, E-selectin associates via its cytoplasmic domain with components of the actin cytoskeleton and undergoes alterations in phosphorylation state that result in changes in gene expression. In this study, we show that E-selectin is localized in cholesterol-rich lipid rafts at the cell surface, and that upon ligation E-selectin clusters and redistributes in the plasma membrane colocalizing with a fraction of caveolin-1-containing rafts. In addition, we demonstrate that leukocyte adhesion via E-selectin results in association with and activation of phospholipase Cgamma (PLCgamma). Moreover, we show that disruption of lipid rafts with the cholesterol-depleting drug methyl-beta-cyclodextrin disrupts the raft localization of E-selectin as well as the ligation-induced association of E-selectin with PLCgamma, and subsequent tyrosine phosphorylation of PLCgamma. In contrast, cholesterol depletion has no effect on E-selectin-dependent mitogen-activated protein kinase activation. Thus, these findings demonstrate that the presence of E-selectin in lipid rafts is necessary for its association with, and activation of, PLCgamma, and suggest that this subcellular localization of E-selectin is related to its signaling function(s) during leukocyte-endothelial interactions.     

Neuronal precursor cell-expressed developmentally down-regulated 4-1 (NEDD4-1) controls the sorting of newly synthesized Ca(V)1.2 calcium channels

Neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) proteins are ubiquitin ligases, which attach ubiquitin moieties to their target proteins, a post-translational modification that is most commonly associated with protein degradation. Nedd4 ubiquitin ligases have been shown to down-regulate both potassium and sodium channels. In this study, we investigated whether Nedd4 ubiquitin ligases also regulate Ca(v) calcium channels. We expressed three Nedd4 family members, Nedd4-1, Nedd4-2, and WWP2, together with Ca(v)1.2 channels in tsA-201 cells. We found that Nedd4-1 dramatically decreased Ca(v) whole-cell currents, whereas Nedd4-2 and WWP2 failed to regulate the current. Surface biotinylation assays revealed that Nedd4-1 decreased the number of channels inserted at the plasma membrane. Western blots also showed a concomitant decrease in the total expression of the channels. Surprisingly, however, neither the Ca(v) pore-forming α1 subunit nor the associated Ca(v)β and Ca(v)α(2)δ subunits were ubiquitylated by Nedd4-1. The proteasome inhibitor MG132 prevented the degradation of Ca(v) channels, whereas monodansylcadaverine and chloroquine partially antagonized the Nedd4-1-induced regulation of Ca(v) currents. Remarkably, the effect of Nedd4-1 was fully prevented by brefeldin A. These data suggest that Nedd4-1 promotes the sorting of newly synthesized Ca(v) channels for degradation by both the proteasome and the lysosome. Most importantly, Nedd4-1-induced regulation required the co-expression of Ca(v)β subunits, known to antagonize the retention of the channels in the endoplasmic reticulum. Altogether, our results suggest that Nedd4-1 interferes with the chaperon role of Ca(v)β at the endoplasmic reticulum/Golgi level to prevent the delivery of Ca(v) channels at the plasma membrane.     

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.     

Proteomics of MUC1‐containing lipid rafts from plasma membranes and exosomes of human breast carcinoma cells MCF‐7

Apically expressed human MUC1 is known to become endocytosed and either to re‐enter the secretory pathway for recycling to the plasma membrane or to be exported by the cells via the formation of multi‐vesicular bodies and the release of exosomes. By using recombinant fusion‐tagged MUC1 as a bait protein we followed an anti‐myc affinity‐based approach for isolating subpopulations of lipid rafts from the plasma membranes and exosomes of MCF‐7 breast cancer cells. MUC1⁺ lipid rafts were not only found to contain genuine raft proteins (flotillin‐1, prohibitin, G protein, annexin A2), but also raft‐associated proteins linking these to the cytoskeleton (ezrin/villin‐2, profilin II, HSP27, γ‐actin, β‐actin) or proteins in complexes with raft proteins, including the bait protein (HSP60, HSP70). Major overlaps were revealed for the subproteomes of plasma membranous and exosomal lipid raft preparations, indicating that MUC1 is sorted into subpopulations of rafts for its trafficking via flotillin‐dependent pathways and export via exosomes.     

TLR4, ethanol, and lipid rafts: a new mechanism of ethanol action with implications for other receptor-mediated effects

Ethanol (EtOH) is the most widely abused substance in the United States, and it contributes to well-documented harmful (at high dosages) and beneficial (at low dosages) changes in inflammatory and immune responses. Lipid rafts have been implicated in the regulation and activation of several important receptor complexes in the immune system, including the TLR4 complex. Many questions remain about the precise mechanisms by which rafts regulate the assembly of these receptor complexes. Results summarized in this review indicate that EtOH acts by altering the LPS-induced redistribution of components of the TLR4 complex within the lipid raft and that this is related to changes in actin cytoskeleton rearrangement, receptor clustering, and subsequent signaling. EtOH provides an example of an immunomodulatory drug that acts at least in part by modifying lipid rafts, and it could represent a model to probe the relationships between rafts, receptor complexes, and signaling.