PSD-95 and Lin-7b interact with acid-sensing ion channel-3 and have opposite effects on H+- gated current

The acid-sensing ion channel-3 (ASIC3) is a degenerin/epithelial sodium channel expressed in the peripheral nervous system. Previous studies indicate that it participates in the response to mechanical and painful stimuli, perhaps contributing to mechanoreceptor and/or H+ -gated nociceptor function. ASIC3 subunits contain intracellular N and C termini that may control channel localization and function. We found that a PDZ-binding motif at the ASIC3 C terminus interacts with four different proteins that contain PDZ domains: PSD-95, Lin-7b, MAGI-1b, and PIST. ASIC3 and these interacting proteins were expressed in dorsal root ganglia and spinal cord, and PSD-95 co-precipitated ASIC3 from spinal cord. When expressed in heterologous cells, PSD-95 reduced the amplitude of ASIC3 acid-evoked currents, whereas Lin-7b increased current amplitude. PSD-95 and Lin-7b altered current density by decreasing or increasing, respectively, the amount of ASIC3 on the cell surface. The finding that multiple PDZ-containing proteins bind ASIC3 and can influence its presence in the plasma membrane suggests that they may play an important role in the contribution of ASIC3 to nociception and mechanosensation.     

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

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

ASIC2 Subunits Facilitate Expression at the Cell Surface and Confer Regulation by PSD-95

Acid-sensing ion channels (ASICs) are Na⁺ channels activated by changes in pH within the peripheral and central nervous systems. Several different isoforms of ASICs combine to form trimeric channels, and their properties are determined by their subunit composition. ASIC2 subunits are widely expressed throughout the brain, where they heteromultimerize with their partnering subunit, ASIC1a. However, ASIC2 contributes little to the pH sensitivity of the channels, and so its function is not well understood. We found that ASIC2 increased cell surface levels of the channel when it is coexpressed with ASIC1a, and genetic deletion of ASIC2 reduced acid-evoked current amplitude in mouse hippocampal neurons. Additionally, ASIC2a interacted with the neuronal synaptic scaffolding protein PSD-95, and PSD-95 reduced cell surface expression and current amplitude in ASICs that contain ASIC2a. Overexpression of PSD-95 also reduced acid-evoked current amplitude in hippocampal neurons. This result was dependent upon ASIC2 since the effect of PSD-95 was abolished in ASIC2−/− neurons. These results lend support to an emerging role of ASIC2 in the targeting of ASICs to surface membranes, and allows for interaction with PSD-95 to regulate these processes.     

PSD-95 and SAP97 exhibit distinct mechanisms for regulating K(+) channel surface expression and clustering

Mechanisms of ion channel clustering by cytoplasmic membrane-associated guanylate kinases such as postsynaptic density 95 (PSD-95) and synapse-associated protein 97 (SAP97) are poorly understood. Here, we investigated the interaction of PSD-95 and SAP97 with voltage-gated or Kv K(+) channels. Using Kv channels with different surface expression properties, we found that clustering by PSD-95 depended on channel cell surface expression. Moreover, PSD-95-induced clusters of Kv1 K(+) channels were present on the cell surface. This was most dramatically demonstrated for Kv1.2 K(+) channels, where surface expression and clustering by PSD-95 were coincidentally promoted by coexpression with cytoplasmic Kvbeta subunits. Consistent with a mechanism of plasma membrane channel-PSD-95 binding, coexpression with PSD-95 did not affect the intrinsic surface expression characteristics of the different Kv channels. In contrast, the interaction of Kv1 channels with SAP97 was independent of Kv1 surface expression, occurred intracellularly, and prevented further biosynthetic trafficking of Kv1 channels. As such, SAP97 binding caused an intracellular accumulation of each Kv1 channel tested, through the accretion of SAP97 channel clusters in large (3-5 microm) ER-derived intracellular membrane vesicles. Together, these data show that ion channel clustering by PSD-95 and SAP97 occurs by distinct mechanisms, and suggests that these channel-clustering proteins may play diverse roles in regulating the abundance and distribution of channels at synapses and other neuronal membrane specializations.     

Neuregulin-1 proteins in rat brain and transfected cells are localized to lipid rafts

Neuregulin-1 proteins and their receptors, which are members of the ErbB subfamily of receptor tyrosine kinases, play essential roles in the development of the nervous system and heart. Most neuregulin-1 isoforms are synthesized as transmembrane proproteins that are proteolytically processed to yield an N-terminal fragment containing the bioactive EGF-like domain. In this study we investigated whether neuregulins are found in lipid rafts, membrane microdomains hypothesized to have important roles in signal transduction, protein trafficking, and proteolytic processing. We found that 45% of a 140-kDa neuregulin protein in rat brain synaptosomal plasma membrane fractions was insoluble in 1% Triton X-100. Flotation gradient analysis demonstrated the presence of the brain 140 kDa neuregulin protein in low-density fractions enriched in PSD-95, a known lipid raft protein. In transfected cells expressing the neuregulin I-beta 1a or the III-beta 1a isoform, most of the neuregulin proprotein was insoluble in 1% Triton X-100, and neuregulin proproteins and C-terminal fragments were detected in lipid raft fractions. In contrast, the III-beta 1a N-terminal fragment was detected only in the detergent-soluble fraction. These results suggest that localization of neuregulins to lipid rafts may play a role in neuregulin signaling within the nervous system.     

Stomatin modulates gating of acid-sensing ion channels

Acid-sensing ion channels (ASICs) are H(+)-gated members of the degenerin/epithelial Na(+) channel (DEG/ENaC) family in vertebrate neurons. Several ASICs are expressed in sensory neurons, where they play a role in responses to nociceptive, taste, and mechanical stimuli; others are expressed in central neurons, where they participate in synaptic plasticity and some forms of learning. Stomatin is an integral membrane protein found in lipid/protein-rich microdomains, and it is believed to regulate the function of ion channels and transporters. In Caenorhabditis elegans, stomatin homologs interact with DEG/ENaC channels, which together are necessary for normal mechanosensation in the worm. Therefore, we asked whether stomatin interacts with and modulates the function of ASICs. We found that stomatin co-immunoprecipitated and co-localized with ASIC proteins in heterologous cells. Moreover, stomatin altered the function of ASIC channels. Stomatin potently reduced acid-evoked currents generated by ASIC3 without changing steady state protein levels or the amount of ASIC3 expressed at the cell surface. In contrast, stomatin accelerated the desensitization rate of ASIC2 and heteromeric ASICs, whereas current amplitude was unaffected. These data suggest that stomatin binds to and alters the gating of ASICs. Our findings indicate that modulation of DEG/ENaC channels by stomatin-like proteins is evolutionarily conserved and may have important implications for mammalian nociception and mechanosensation.     

Association of excitatory amino acid transporters, especially EAAT2, with cholesterol-rich lipid raft microdomains: importance for excitatory amino acid transporter localization and function

In the present study, we investigated the role of membrane cholesterol in the function of glutamate transporters. Depletion of membrane cholesterol by methyl-beta-cyclodextrin resulted in reduced Na(+)-dependent glutamate uptake in primary cortical cultures. Glial glutamate transporter EAAT2-mediated uptake was more sensitive to this effect. Cell surface biotinylation and immunostaining experiments revealed that the loss of cholesterol significantly altered the trafficking of EAAT2 to the plasma membrane as well as their membrane distribution. These effects were also observed in neuronal glutamate transporter EAAT3 but to a lesser extent. Furthermore, the treatment of mouse brain plasma membrane vesicles with methyl-beta-cyclodextrin resulted in a significant reduction in glutamate uptake, suggesting that cholesterol depletion has a direct effect on the function of the glutamate transporters. Plasma membrane cholesterol is localized within discreet microdomains known as lipid rafts. Analyses of purified lipid raft microdomains revealed that a large portion of total EAAT2 and a minor portion of total EAAT1, EAAT3, and EAAT4 were associated with lipid rafts. Artificial aggregation of lipid rafts in vivo resulted in the formation of larger EAAT2-immunoreactive clusters on the cell surface. The purified lipid raft-associated fractions were capable of Na(+)-dependent glutamate uptake. Our data suggest that the glutamate transporters, especially EAAT2, are associated with cholesterol-rich lipid raft microdomains of the plasma membrane and that the association with these cholesterol-rich microdomains is important for excitatory amino acid transporter localization and function.     

Isoform-specific localization of voltage-gated K+ channels to distinct lipid raft populations. Targeting of Kv1.5 to caveolae

The precise subcellular localization of ion channels is often necessary to ensure rapid and efficient integration of both intracellular and extracellular signaling events. Recently, we have identified lipid raft association as a novel mechanism for the subcellular sorting of specific voltage-gated K(+) channels to regions of the membrane rich in signaling complexes. Here, we demonstrate isoform-specific targeting of voltage-gated K(+) (Kv) channels to distinct lipid raft populations with the finding that Kv1.5 specifically targets to caveolae. Multiple lines of evidence indicate that Kv1.5 and Kv2.1 exist in distinct raft domains: 1) channel/raft association shows differential sensitivity to increasing concentrations of Triton X-100; 2) unlike Kv2.1, Kv1.5 colocalizes with caveolin on the cell surface and redistributes with caveolin following microtubule disruption; and 3) immunoisolation of caveolae copurifies Kv1.5 channel. Both depletion of cellular cholesterol and inhibition of sphingolipid synthesis alter Kv1.5 channel function by inducing a hyperpolarizing shift in the voltage dependence of activation and inactivation. The differential targeting of Kv channel subtypes to caveolar and noncaveolar rafts within a single membrane represents a unique mechanism of compartmentalization, which may permit isoform-specific modulation of K(+) channel function.     

Acid-sensing ion channel 2 (ASIC2) modulates ASIC1 H+-activated currents in hippocampal neurons

Hippocampal neurons express subunits of the acid-sensing ion channel (ASIC1 and ASIC2) and exhibit large cation currents that are transiently activated by acidic extracellular solutions. Earlier work indicated that ASIC1 contributed to the current in these neurons and suggested its importance for normal behavior. However, the specific contribution of ASIC1 and ASIC2 subunits to acid-evoked currents in hippocampal neurons remained uncertain. To decipher the individual role of the ASIC subunits, we studied H(+)-gated currents in neurons from both ASIC1 and ASIC2 null mice. We found that much of the current was produced by ASIC1a/2a heteromultimeric channels, and individual subunits made distinct contributions. The ASIC1a subunit was key in establishing current amplitude. The ASIC2a subunit had little effect on amplitude but influenced desensitization, recovery from desensitization, pH sensitivity, and the response to modulatory agents. We also found heterogeneity in the contribution of ASIC2 throughout the neuronal population, with individual neurons expressing both ASIC1a homomultimeric and ASIC1a/2a heteromultimeric channels. Studies of neurons heterozygous for disrupted ASIC alleles indicated that the properties of H(+)-gated currents are dependent on the proportion of the individual subunits. These findings indicate that the absolute and relative amounts of ASIC subunits determine the amplitude and properties of hippocampal H(+)-gated currents and therefore may contribute to normal physiology and pathophysiology.     

The multivalent PDZ domain-containing protein CIPP is a partner of acid-sensing ion channel 3 in sensory neurons

Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular pH. They are present in the brain, where they are thought to participate in signal transduction associated with local pH variations, and in sensory neurons, where they have been involved in pain perception associated with tissue acidosis and in mechanoperception. The ASIC3 subunit is mainly expressed in dorsal root ganglion neurons. Its expression is associated with a rapidly inactivating current followed by a slowly activating sustained current thought to be required for the tonic sensation of pain caused by acids. We report here the interaction of this channel subunit with the multivalent PDZ (PSD-95 Drosophila discs-large protein, Zonula occludens protein 1) domain-containing protein CIPP. This interaction requires the C-terminal region of ASIC3 and the fourth PDZ domain of CIPP. Co-expression of CIPP and ASIC3 in COS cells increases the maximal ASIC3 peak current density by a factor of 5 and slightly shifts the pH(0.5) for activation from pH 6.2 to pH 6.4. CIPP mRNA is found at a significant level in the same dorsal root ganglion neuronal cell population that expresses the ASIC3 subunit, i.e. mainly in the small nociceptive neurons. CIPP is thus a scaffolding protein that could both enhance the surface expression of ASIC3 and bring together ASIC3 and functionally related proteins in the membrane of sensory neurons.     

Lipid raft localization of GABA A receptor and Na+, K+-ATPase in discrete microdomain clusters in rat cerebellar granule cells

The microdomain localization of the GABA(A) receptor in rat cerebellar granule cells was studied by subcellular fractionation and fluorescence- and immunogold electron microscopy. The receptor resided in lipid rafts, prepared at 37 degrees C by extraction with the nonionic detergent Brij 98, but the raft fraction, defined by the marker ganglioside GM(1) in the floating fractions following density gradient centrifugation, was heterogeneous in density and protein composition. Thus, another major raft-associated membrane protein, the Na(+), K(+)-ATPase, was found in discrete rafts of lower density, reflecting clustering of the two proteins in separate membrane microdomains. Both proteins were observed in patchy "hot spots" at the cell surface as well as in isolated lipid rafts. Their insolubility in Brij 98 was only marginally affected by methyl-beta-cyclodextrin. In contrast, both the GABA(A) receptor and Na(+), K(+)-ATPase were largely soluble in ice cold Triton X-100. This indicates that Brij 98 extraction defines an unusual type of cholesterol-independent lipid rafts that harbour membrane proteins also associated with underlying scaffolding/cytoskeletal proteins such as gephyrin (GABA(A) receptor) and ankyrin G (Na(+), K(+)-ATPase). By providing an ordered membrane microenvironment, lipid rafts may contribute to the clustering of the GABA(A) receptor and the Na(+), K(+)-ATPase at distinct functional locations on the cell surface.     

Role of lipid rafts in membrane delivery of renal epithelial Na+-K+-ATPase, thick ascending limb

Lipid rafts are cholesterol- and shingolipid-enriched membrane microdomains implicated in membrane signaling and trafficking. To assess renal epithelial raft functions through the characterization of their associated membrane proteins, we have isolated lipid rafts from rat kidney by sucrose gradient fractionation after detergent treatment. The low-density fraction was enriched in cholesterol, sphingolipid, and flotillin-1 known as lipid raft markers. Based on proteomic analysis of the low-density fraction, the protein with the highest significance score was the alpha-subunit of Na(+)-K(+)-ATPase (NKA), whose raft association was validated by simultaneous immunoblotting. The beta-subunit of NKA was copurified from the low-density fraction. To test the role of lipid rafts in sorting and membrane delivery of renal-transporting epithelia, we have chosen to study thick ascending limb (TAL) epithelium for its high NKA activity and the property to be stimulated by antidiuretic hormone (ADH). Cultured rabbit TAL cells were studied. Cholesterol depletion and detergent extraction at warmth caused a shift of NKA to the higher-density fractions. Comparative preparations from blood monocytes revealed the absence of NKA from rafts in these nonpolarized cells. Short-term exposure of rabbit TAL cells to ADH (1 h) caused translocation and enhanced raft association of NKA via cAMP activation. Preceding cholesterol depletion prevented this effect. TAL-specific, glycosylphosphatidylinositol-anchored Tamm Horsfall protein was copurified with NKA in the same raft fraction, suggesting functional interference between these products. These results may have functional implications regarding the turnover, trafficking, and regulated surface expression of NKA as the major basolateral ion transporter of TAL.     

Vaccinia virus penetration requires cholesterol and results in specific viral envelope proteins associated with lipid rafts

Vaccinia virus infects a wide variety of mammalian cells from different hosts, but the mechanism of virus entry is not clearly defined. The mature intracellular vaccinia virus contains several envelope proteins mediating virion adsorption to cell surface glycosaminoglycans; however, it is not known how the bound virions initiate virion penetration into cells. For this study, we investigated the importance of plasma membrane lipid rafts in the mature intracellular vaccinia virus infection process by using biochemical and fluorescence imaging techniques. A raft-disrupting drug, methyl-beta-cyclodextrin, inhibited vaccinia virus uncoating without affecting virion attachment, indicating that cholesterol-containing lipid rafts are essential for virion penetration into mammalian cells. To provide direct evidence of a virus and lipid raft association, we isolated detergent-insoluble glycolipid-enriched membranes from cells immediately after virus infection and demonstrated that several viral envelope proteins, A14, A17L, and D8L, were present in the cell membrane lipid raft fractions, whereas the envelope H3L protein was not. Such an association did not occur after virions attached to cells at 4 degrees C and was only observed when virion penetration occurred at 37 degrees C. Immunofluorescence microscopy also revealed that cell surface staining of viral envelope proteins was colocalized with GM1, a lipid raft marker on the plasma membrane, consistent with biochemical analyses. Finally, mutant viruses lacking the H3L, D8L, or A27L protein remained associated with lipid rafts, indicating that the initial attachment of vaccinia virions through glycosaminoglycans is not required for lipid raft formation.     

Free cholesterol alters lipid raft structure and function regulating neutrophil Ca2+ entry and respiratory burst: correlations with calcium channel raft trafficking

Recent studies associate cholesterol excess and atherosclerosis with inflammation. The link between these processes is not understood, but cholesterol is an important component of lipid rafts. Rafts are thought to concentrate membrane signaling molecules and thus regulate cell signaling through G protein-coupled pathways. We used methyl beta-cyclodextrin to deplete cholesterol from polymorphonuclear neutrophil (PMN) rafts and thus study the effects of raft disruption on G protein-coupled Ca(2+) mobilization. Methyl beta-cyclodextrin had no effect on Ca(2+) store depletion by the G protein-coupled agonists platelet-activating factor or fMLP, but abolished agonist-stimulated Ca(2+) entry. Free cholesterol at very low concentrations regulated Ca(2+) entry into PMN via nonspecific Ca(2+) channels in a biphasic fashion. The specificity of cholesterol regulation for Ca(2+) entry was confirmed using thapsigargin studies. Responses to cholesterol appear physiologic because they regulate respiratory burst in a proportional biphasic fashion. Investigating further, we found that free cholesterol accumulated in PMN lipid raft fractions, promoting formation and polarization of membrane rafts. Finally, the transient receptor potential calcium channel protein TRPC1 redistributed to raft fractions in response to cholesterol. The uniformly biphasic relationships between cholesterol availability, Ca(2+) signaling and respiratory burst suggest that Ca(2+) influx and PMN activation are regulated by the quantitative relationships between cholesterol and other environmental lipid raft components. The association between symptomatic cholesterol excess and inflammation may therefore in part reflect free cholesterol- dependent changes in lipid raft structure that regulate immune cell Ca(2+) entry. Ca(2+) entry-dependent responses in other cell types may also reflect cholesterol bioavailability and lipid incorporation into rafts.     

Multiple Kv1.5 targeting to membrane surface microdomains

Surface expression of voltage-dependent K(+) channels (Kv) has a pivotal role in leukocyte physiology. Although little is known about the physiological role of lipid rafts, these microdomains concentrate signaling molecules and their ion channel substrates. Kv1.3 associates with Kv1.5 to form functional channels in macrophages. Different isoform stoichiometries lead to distinct heteromeric channels which may be further modulated by targeting the complex to different membrane surface microdomains. Kv1.3 targets to lipid rafts, whereas Kv1.5 localization is under debate. With this in mind, we wanted to study whether heterotetrameric Kv1.5-containing channels target to lipid rafts. While in transfected HEK-293 cells, homo- and heterotetrameric channels targeted to rafts, Kv1.5 did not target to rafts in macrophages. Therefore, Kv1.3/Kv1.5 hybrid channels are mostly concentrated in non-raft microdomains. However, LPS-induced activation, which increases the Kv1.3/Kv1.5 ratio and caveolin, targeted Kv1.5 back to lipid rafts. Moreover, Kv1.5 did not localize to low-buoyancy fractions in L6E9 skeletal myoblasts, which also coexpress both channels, heart membranes or cardiomyocyes. Coexpression of a Cav3(DGV)-mutant confined Kv1.5 to Cav3(DGV)-vesicles of HEK cells. Contrarily, coexpression of Kvbeta2.1 impaired the Kv1.5 targeting to raft microdomains in HEK cells. Our results indicate that Kv1.5 partnership interactions are underlying mechanisms governing channel targeting to lipid rafts.     

The acid-activated ion channel ASIC contributes to synaptic plasticity, learning, and memory

Many central neurons possess large acid-activated currents, yet their molecular identity is unknown. We found that eliminating the acid sensing ion channel (ASIC) abolished H(+)-gated currents in hippocampal neurons. Neuronal H(+)-gated currents and transient acidification are proposed to play a role in synaptic transmission. Investigating this possibility, we found ASIC in hippocampus, in synaptosomes, and in dendrites localized at synapses. Moreover, loss of ASIC impaired hippocampal long-term potentiation. ASIC null mice had reduced excitatory postsynaptic potentials and NMDA receptor activation during high-frequency stimulation. Consistent with these findings, null mice displayed defective spatial learning and eyeblink conditioning. These results identify ASIC as a key component of acid-activated currents and implicate these currents in processes underlying synaptic plasticity, learning, and memory.     

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.     

Differential targeting of Shaker-like potassium channels to lipid rafts

Ion channel targeting within neuronal and muscle membranes is an important determinant of electrical excitability. Recent evidence suggests that there exists within the membrane specialized microdomains commonly referred to as lipid rafts. These domains are enriched in cholesterol and sphingolipids and concentrate a number of signal transduction proteins such as nitric-oxide synthase, ligand-gated receptors, and multiple protein kinases. Here, we demonstrate that the voltage-gated K(+) channel Kv2.1, but not Kv4.2, targets to lipid rafts in both heterologous expression systems and rat brain. The Kv2.1 association with lipid rafts does not appear to involve caveolin. Depletion of cellular cholesterol alters the buoyancy of the Kv2.1 associated rafts and shifts the midpoint of Kv2.1 inactivation by nearly 40 mV without affecting peak current density or channel activation. The differential targeting of Kv channels to lipid rafts represents a novel mechanism both for the subcellular sorting of K(+) channels to regions of the membrane rich in signaling complexes and for modulating channel properties via alterations in lipid content.     

Phosphoinositide 3-kinase-independent non-genomic signals transit from the androgen receptor to Akt1 in membrane raft microdomains

The serine-threonine kinase, Akt1/protein kinase Balpha is an important mediator of growth, survival, and metabolic signaling. Recent studies have implicated cholesterol-rich, lipid raft microdomains in survival signals mediated by Akt1. Here we address the role of lipid raft membranes as a potential site of intersection of androgenic and Akt1 signaling. A subpopulation of androgen receptor (AR) was found to localize to a lipid raft subcellular compartment in LNCaP prostate cancer cells. Endogenous AR interacted with endogenous Akt1 preferentially in lipid raft fractions and androgen substantially enhanced the interaction between the two proteins. The association of AR with Akt1 was inhibited by the anti-androgen, bicalutamide, but was not affected by inhibition of phosphoinositide 3-kinase (PI3K). Androgen promoted endogenous Akt1 activity in lipid raft fractions, in a PI3K-independent manner, within 10 min of treatment. Fusion of a lipid raft targeting sequence to AR enhanced localization of the receptor to rafts, and stimulated Akt1 activity in response to androgen, while reducing the cells' dependence on constitutive signaling through PI3K for cell survival. These findings suggest that signals channeled through AR and Akt1 intersect by a mechanism involving formation within lipid raft membranes of an androgen-responsive, extranuclear AR/Akt1 complex. Our results indicate that cholesterol-rich membrane microdomains play a role in transmitting non-genomic signals involving androgen and the Akt pathway in prostate cancer cells.