Detergent-soluble Fraction Research Articles

STT3B-Dependent Posttranslational N-Glycosylation as a Surveillance System for Secretory Protein

Nascent secretory proteins are extensively scrutinized at the endoplasmic reticulum (ER). Various signatures of client proteins, including exposure of hydrophobic patches or unpaired sulfhydryls, are coordinately utilized to reduce nonnative proteins in the ER. We report here the cryptic N-glycosylation site as a recognition signal for unfolding of a natively nonglycosylated protein, transthyretin (TTR), involved in familial amyloidosis. Folding and ER-associated degradation (ERAD) perturbation analyses revealed that prolonged TTR unfolding induces externalization of cryptic N-glycosylation site and triggers STT3B-dependent posttranslational N-glycosylation. Inhibition of posttranslational N-glycosylation increases detergent-insoluble TTR aggregates and decreases cell proliferation of mutant TTR-expressing cells. Moreover, this modification provides an alternative pathway for degradation, which is EDEM3-mediated N-glycan-dependent ERAD, distinct from the major pathway of Herp-mediated N-glycan-independent ERAD. Hence we postulate that STT3B-dependent posttranslational N-glycosylation is part of a triage-salvage system recognizing cryptic N-glycosylation sites of secretory proteins to preserve protein homeostasis.

Membrane events after cross-linking of the chB6 alloantigen (111.42)

Abstract Chickens are one of the classic models of vertebrate immunity. We have been interested in the role of the alloantigen chB6 (formerly called BU1) in the development of B cells within the bursa and have presented evidence that chB6 can trigger apoptosis. Furthermore, the pathway that triggers apoptosis is similar to that triggered by FAS, with caspase 8 as an initiator, caspases 9 and 3 as later caspases, and bcl-xl functioning as an inhibitor of apoptosis. We have been unable to demonstrate a steady state association of chB6 and caspase 8 in DT40, so the mechanism of caspase 8 activation remains unclear. Here we show that chB6 localizes in distinct clusters on the surface of DT40 cells, reminiscent of the SPOTS others have described in Fas signaling. These clusters co-localize with cholera toxin labeled lipid rafts and chB6 appears to be internalized by the B cells. Western blots indicate that chB6 is present in both detergent soluble and insoluble membrane fractions on resting DT 40 cells. However, cross-linking chB6 with a secondary antibody shows that chB6 is internalized selectively from detergent insoluble (lipid raft) fractions of the cell membrane. This suggests an active distribution of chB6 in the membrane and an internal trafficking that may be functionally important. This work offers a new insight into the signaling via chB6. This work supported by a grant from the DePaul University Research Council.

Corticotropin-releasing factor receptor-dependent effects of repeated stress on tau phosphorylation, solubility, and aggregation

Exposure and/or sensitivity to stress have been implicated as conferring risk for development of Alzheimer's disease (AD). Although the basis for such a link remains unclear, we previously reported differential involvement of corticotropin-releasing factor receptor (CRFR) 1 and 2 in acute stress-induced tau phosphorylation (tau-P) and solubility in the hippocampus. Here we examined the role of CRFRs in tau-P induced by repeated stress and the structural manifestations of altered tau solubility. Robust tau-P responses were seen in WT and CRFR2 null mice exposed to repeated stress, which were sustained at even 24 h after the final stress exposure. A portion of phosphorylated tau in these mice was sequestered in detergent-soluble cellular fractions. In contrast, CRFR1 and CRFR double-KO mice did not exhibit repeated stress-induced alterations in tau-P or solubility. Similarly, treatment with CRFR1 antagonist attenuated repeated stress-induced tau-P. Using histochemical approaches in a transgenic CRFR1 reporter mouse line, we found substantial overlap between hippocampal CRFR1 expression and cells positive for phosphorylated tau after exposure to repeated stress. Ultrastructural analysis of negatively stained extracts from WT and CRFR2 null mice identified globular aggregates that displayed positive immunogold labeling for tau-P, as well as conformational changes in tau (MC1) seen in early AD. Given that repeated stress exposure results in chronic increases in hippocampal tau-P and its sequestration in an insoluble (and potentially prepathogenic) form, our data may define a link between stress and an AD-related pathogenic mechanism.

Identification of Novel Interaction between Annexin A2 and Keratin 17: EVIDENCE FOR RECIPROCAL REGULATION

Keratins are cytoplasmic intermediate filament proteins providing crucial structural support in epithelial cells. Keratin expression has diagnostic and even prognostic value in disease settings, and recent studies have uncovered modulatory roles for select keratin proteins in signaling pathways regulating cell growth and cell death. Elevated keratin expression in select cancers is correlated with higher expression of EGF receptor (EGFR), whose overexpression and/or mutation give rise to cancer. To explore the role of keratins in oncogenic signaling pathways, we examined the regulation of epithelial growth-associated keratin 17 (K17) in response to EGFR activation. K17 is specifically up-regulated in detergent-soluble fraction upon EGFR activation, and immunofluorescence analysis revealed alterations in K17-containing filaments. Interestingly, we identified AnxA2 as a novel interacting partner of K17, and this interaction is antagonized by EGFR activation. K17 and AnxA2 proteins show reciprocal regulation. Modulating expression of AnxA2 altered K17 stability, and AnxA2 overexpression delays EGFR-mediated change in K17 detergent solubility. Down-regulation of K17 expression, in turn, results in decreased AnxA2 phosphorylation at Tyr-23. These findings uncover a novel interaction involving K17 and AnxA2 and identify AnxA2 as a potential regulator of keratin filaments.

Cholesterol dependence of Newcastle Disease Virus entry

Lipid rafts are membrane microdomains enriched in cholesterol, sphingolipids, and glycolipids that have been implicated in many biological processes. Since cholesterol is known to play a key role in the entry of some other viruses, we investigated the role of cholesterol and lipid rafts in the host cell plasma membrane in Newcastle Disease Virus (NDV) entry. We used methyl-β-cyclodextrin (MβCD) to deplete cellular cholesterol and disrupt lipid rafts. Our results show that the removal of cellular cholesterol partially reduces viral binding, fusion and infectivity. MβCD had no effect on the expression of sialic acid containing molecule expression, the NDV receptors in the target cell. All the above-described effects were reversed by restoring cholesterol levels in the target cell membrane. The HN viral attachment protein partially localized to detergent-resistant membrane microdomains (DRMs) at 4°C and then shifted to detergent-soluble fractions at 37°C. These results indicate that cellular cholesterol may be required for optimal cell entry in NDV infection cycle.

Desmoplakin is Important for Proper Cardiac Cell-Cell Interactions

Normal cardiac function is maintained through dynamic interactions of cardiac cells with each other and with the extracellular matrix. These interactions are important for remodeling during cardiac growth and pathophysiological conditions. However, the precise mechanisms of these interactions remain unclear. In this study we examined the importance of desmoplakin (DSP) in cardiac cell-cell interactions. Cell-cell communication in the heart requires the formation and preservation of cell contacts by cell adhesion junctions called desmosome-like structures. A major protein component of this complex is DSP, which plays a role in linking the cytoskeletal network to the plasma membrane. Our laboratory previously generated a polyclonal antibody (1611) against the detergent soluble fraction of cardiac fibroblast plasma membrane. In attempting to define which proteins 1611 recognizes, we performed two-dimensional electrophoresis and identified DSP as one of the major proteins recognized by 1611. Immunoprecipitation studies demonstrated that 1611 was able to directly pulldown DSP. We also demonstrate that 1611 and anti-DSP antibodies co-localize in whole heart sections. Finally, using a three-dimensional in vitro cell-cell interaction assay, we demonstrate that 1611 can inhibit cell-cell interactions. These data indicate that DSP is an important protein for cell-cell interactions and affects a variety of cellular functions, including cytokine secretion.

Characterization of glyceraldehyde-3-phosphate dehydrogenase as a novel transferrin receptor

A majority of cells obtain of transferrin (Tf) bound iron via transferrin receptor 1 (TfR1) or by transferrin receptor 2 (TfR2) in hepatocytes. Our study establishes that cells are capable of acquiring transferrin iron by an alternate pathway via GAPDH.These findings demonstrate that upon iron depletion, GAPDH functions as a preferred receptor for transferrin rather than TfR1 in some but not all cell types. We utilized CHO-TRVb cells that do not express TfR1 or TfR2 as a model system. A knockdown of GAPDH in these cells resulted in a decrease of not only transferrin binding but also associated iron uptake. The current study also demonstrates that, unlike TfR1 and TfR2 which are localized to a specific membrane fraction, GAPDH is located in both the detergent soluble and lipid raft fractions of the cell membrane. Further, transferrin uptake by GAPDH occurs by more than one mechanism namely clathrin mediated endocytosis, lipid raft endocytosis and macropinocytosis. By determining the kinetics of this pathway it appears that GAPDH–Tf uptake is a low affinity, high capacity, recycling pathway wherein transferrin is catabolised. Our findings provide an explanation for the detailed role of GAPDH mediated transferrin uptake as an alternate route by which cells acquire iron.

Dissociation of actin polymerization and lipid raft accumulation by ligation of the Inducible Costimulator (ICOS, CD278)

T lymphocyte antigen activation is facilitated by clustering of membrane glycosphingolipid-enriched microdomains (GEMs, lipid “rafts”) at the T cell/APC contact that is linked to changes in actin cytoskeleton and is one major mechanism of CD28 costimulation. Ligation of CD28 alone, or ligation of the CD28-like molecules CTLA-4 (CD152) and ICOS (CD278) induces actin polymerization with cell elongation and generation of lamellipodia and filopodia in T cells. These changes are dependent on Src, PI3-kinase, Vav, and Rho family GTPases. Whereas CD28 and CTLA-4 have been shown to be functional and physically associated with lipid rafts, the presence of ICOS in lipid rafts or its effect in raft clustering is not known. In this work, we have activated the T cell line D10 with anti-ICOS antibodies, alone or combined with anti-CD3 antibodies, bound or unbound to polystyrene microbeads or glass coverslips. The possible relationship of ICOS-induced changes in actin cytoskeleton to the ICOS localization in membrane rafts was then analyzed by fluorescence microscopy, or by immunoblot of detergent insoluble (“raft”) or soluble (“non-raft”) fractions of cell lysates. Our data show that ICOS promotes TCR/CD3 induction of raft clustering at the site of activation. However, ICOS, which, on its own, can induce accumulations of polymerized actin, is undetectable in membrane rafts, even when using CD3 or ICOS, ligands capable of inducing clear changes in the actin cytoskeleton.

Distribution of C and N in soluble fractionations for characterizing the respective biodegradation of sludge and bulking agents

This study utilized C and N distribution in different soluble fractionations instead of the routine C/N ratio to characterize the respective biodegradation of sludge and bulking agents in bio-drying or composting. For sludge, C was mainly distributed (31.8%) in the neutral detergent soluble and water insoluble fraction (SOL), whereas it was mainly distributed in the cellulose-like fraction (CEL) for straw (39.5%) and sawdust (45.8%). A large proportion of N was in the 35°C water-soluble fraction (W35°C) for sludge (34.0%) and straw (52.5%), while for sawdust it was in the lignin-like fraction (LIG; 49.4%). For sludge, the C and N loss were mainly contributed by W35°C (36.9% and 52.4%). The other fractions also contributed a lot. For straw, 22.4% of C and 89.8% of N lose in W35°C. The hemicellulose-like (HEM) and CEL fraction also gave a large contribution to C loss (28.5% and 40.1%), while contributing little to N loss.

Lamin A and lamin C form homodimers and coexist in higher complex forms both in the nucleoplasmic fraction and in the lamina of cultured human cells

We have investigated and quantified the nuclear A-type lamin pool from human HeLa S3 suspension cells with respect to their distribution to detergent soluble and insoluble fractions. We devised a sequential extraction protocol and found that maximally 10% of A-type lamins are recovered in the soluble fraction. Notably, lamin C is enriched in low detergent fractions and only with 0.5% Nonidet P-40 lamin A and C are recovered in ratios nearly equivalent to those found in whole cell extracts and in the lamina fraction. Authentic nucleoplasmic proteins such as LAP2a, pRB and p53 are co-extracted to a large part together with the A-type lamins in these fractions. By sucrose density centrifugation we revealed that the majority of lamins co-sedimented with human IgG indicating they form rather small complexes in the range of dimers and slightly larger complexes. Some lamin A - but not lamin C - is obtained in addition in a much faster sedimenting fraction. Authentic nuclear proteins such as PCNA, p53 and LAP2a were found both in the light and the heavy sucrose fractions together with lamin A. Last but not least, immunoprecipitation experiments from both soluble fractions and from RIPA lysates of whole cells revealed that lamin A and lamin C do not form heterodimers but segregate practically completely. Correspondingly, immunofluorescence microscopy of formaldehyde-fixed cells clearly demonstrated that lamin A and C are localized at least in part to distinct patches within the lamina. Hence, the structural segregation of lamin A and C is indeed retained in the nuclear envelope to some extent too.

Silencer of Death Domains (SODD) Inhibits Skeletal Muscle and Kidney Enriched Inositol 5-Phosphatase (SKIP) and Regulates Phosphoinositide 3-Kinase (PI3K)/Akt Signaling to the Actin Cytoskeleton

Phosphoinositide 3-kinase (PI3K) regulates cell polarity and migration by generating phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) at the leading edge of migrating cells. The serine-threonine protein kinase Akt binds to PI(3,4,5)P(3), resulting in its activation. Active Akt promotes spatially regulated actin cytoskeletal remodeling and thereby directed cell migration. The inositol polyphosphate 5-phosphatases (5-ptases) degrade PI(3,4,5)P(3) to form PI(3,4)P(2), which leads to diminished Akt activation. Several 5-ptases, including SKIP and SHIP2, inhibit actin cytoskeletal reorganization by opposing PI3K/Akt signaling. In this current study, we identify a molecular co-chaperone termed silencer of death domains (SODD/BAG4) that forms a complex with several 5-ptase family members, including SKIP, SHIP1, and SHIP2. The interaction between SODD and SKIP exerts an inhibitory effect on SKIP PI(3,4,5)P(3) 5-ptase catalytic activity and consequently enhances the recruitment of PI(3,4,5)P(3)-effectors to the plasma membrane. In contrast, SODD(-/-) mouse embryonic fibroblasts exhibit reduced Akt-Ser(473) and -Thr(308) phosphorylation following EGF stimulation, associated with increased SKIP PI(3,4,5)P(3)-5-ptase activity. SODD(-/-) mouse embryonic fibroblasts exhibit decreased EGF-stimulated F-actin stress fibers, lamellipodia, and focal adhesion complexity, a phenotype that is rescued by the expression of constitutively active Akt1. Furthermore, reduced cell migration was observed in SODD(-/-) macrophages, which express the three 5-ptases shown to interact with SODD (SKIP, SHIP1, and SHIP2). Therefore, this study identifies SODD as a novel regulator of PI3K/Akt signaling to the actin cytoskeleton.

Different β-amyloid oligomer assemblies in Alzheimer brains correlate with age of disease onset and impaired cholinergic activity

In this study, we examined the relationship between various β-amyloid (Aβ) oligomer assemblies in autopsy brain with the levels of fibrillar Aβ and cholinergic synaptic function. Brain tissues obtained from the frontal cortex of 14 Alzheimer's disease (AD) patients grouped into early-onset AD (EOAD) and late-onset AD (LOAD) and 12 age-matched control subjects were used to extract and quantify Aβ oligomers in soluble (TBS), detergent soluble (TBST), and insoluble (GuHCl) fractions. The predominant oligomeric Aβ assemblies detected were dodecamers, decamers, and pentamers, and different patterns of expression were observed between EOAD and LOAD patients. There was no association between any of the detected Aβ oligomer assemblies and fibrillar Aβ levels measured by N-methyl[3H] 2-(40-methylaminophenyl)-6-hydroxy-benzothiazole ([3H]PIB) binding. Levels of pentamers in the soluble fraction significantly correlated with a reduction in choline acetyltransferase activity in AD patients. The number of nicotinic acetylcholine receptors negatively correlated with the total amount of Aβ oligomers in the insoluble fraction in EOAD patients, and with decamers in the soluble fraction in LOAD patients. These novel findings suggest that distinct Aβ oligomers induce impairment of cholinergic neurotransmission in AD pathogenesis.

Effect of Shengmai-san on Cognitive Performance and Cerebral Oxidative Damage in BALB/c Mice

The aim of this study was to examine the effect of Shengmai-san (SMS) on learning and memory impairment induced by scopolamine (1 mg/kg, i.p.) in mice. The passive avoidance task (PAT) and Morris water maze (MWM) test served as the behavioral models for testing memory. To elucidate the mechanism of its cognitive-enhancing activity, the effects of SMS (2, 4, or 8 g/kg) on activities of acetylcholinesterase (AChE) and antioxidant enzymes and levels of acetylcholine (ACh) and nitrite were evaluated in brain homogenate. Tacrine (THA) (10 mg/kg, p.o.) was used as a reference drug. SMS (4 or 8 g/kg) significantly prevented scopolamine-induced impairments as measured by the PAT and MWM (probe trial session). SMS (4 or 8 g/kg) also significantly reduced the oxidative-nitrative stress, as evidenced by decreased malondialdehyde and nitrite levels and by its prevention of decreases in glutathione and superoxide dismutase levels. The activity of AChE was decreased in scopolamine-treated mice but was inhibited significantly by SMS treatment (4 or 8 g/kg) in both salt- and detergent-soluble fractions of brain homogenates. Further SMS treatment (4 or 8 g/kg) significantly increased the ACh levels in the brain homogenate to a level similar to that observed in THA treatment. Thus, the significant cognitive enhancement observed after 7 days of administration of SMS is closely related to the strong antioxidant properties of SMS in addition to its inhibition of brain AChE activity. These findings stress the critical impact of SMS on higher brain functions such as learning and memory.

Seroreactivity Against Aqueous-Soluble and Detergent-Soluble Retinal Proteins in Posterior Uveitis

To characterize the seroreactivity against retinal proteins in patients with posterior uveitis, retinal disease of noninflammatory origin, and healthy controls. Patients with posterior uveitis (n=47), molecularly confirmed photoreceptor degenerations (n=11), and healthy controls (n=33) received dilated fundus examinations at the University of Iowa. Aqueous-soluble and detergent-soluble fractions of human retina were separated by gel electrophoresis and transferred to polyvinylidene fluoride membranes. Membranes were probed with patient serum samples to detect IgG, IgA, and IgM human antibodies that react with retinal antigens. The number of bands detected by Western blot was counted, and their molecular weights were determined. Antibodies recognizing retinal proteins were found in healthy controls, in patients with posterior uveitis, and in patients with molecularly confirmed heritable retinal degenerations. In healthy controls, 42% of individuals had circulating autoantibodies that recognized retinal proteins. Healthy controls had a low odds ratio of serum reactivity to soluble antigens (0.7; 95% confidence interval [CI], 0.4-1.2). Patients with inflammatory retinal diseases and inherited retinal diseases had 4.89 (95% CI, 2.25-10.64; P<.001) and 2.71 (95% CI, 1.19-6.16; P=.02) times more activity against soluble retinal antigens compared with controls. Healthy control patients exhibited a significantly higher level of background autoantibody activity against retinal proteins than previously reported. Antibody activity in healthy controls was primarily directed against membrane-bound retinal proteins, whereas in patients with pathologic retinal conditions, antibodies targeting nonmembrane-bound retinal proteins predominate.

In TNF-stimulated Cells, RIPK1 Promotes Cell Survival by Stabilizing TRAF2 and cIAP1, which Limits Induction of Non-canonical NF-κB and Activation of Caspase-8

RIPK1 is involved in signaling from TNF and TLR family receptors. After receptor ligation, RIPK1 not only modulates activation of both canonical and NIK-dependent NF-κB, but also regulates caspase-8 activation and cell death. Although overexpression of RIPK1 can cause caspase-8-dependent cell death, when RIPK1(-/-) cells are exposed to TNF and low doses of cycloheximide, they die more readily than wild-type cells, indicating RIPK1 has pro-survival as well as pro-apoptotic activities. To determine how RIPK1 promotes cell survival, we compared wild-type and RIPK1(-/-) cells treated with TNF. Although TRAF2 levels remained constant in TNF-treated wild-type cells, TNF stimulation of RIPK1(-/-) cells caused TRAF2 and cIAP1 to be rapidly degraded by the proteasome, which led to an increase in NIK levels. This resulted in processing of p100 NF-κB2 to p52, a decrease in levels of cFLIP(L), and activation of caspase-8, culminating in cell death. Therefore, the pro-survival effect of RIPK1 is mediated by stabilization of TRAF2 and cIAP1.

Biochemical characteristics of solid fractions from animal slurry separation and their effects on C and N mineralisation in soil

Solid fractions from separated animal slurry can be used as organic fertilisers on agricultural land. Solid fractions contain variable amounts of inorganic and organic N, so it is important to synchronise their application in the field with crop demand to ensure N availability in the growing season. This study quantified C and N mineralisation for a wide range of solid fractions from slurry separation applied to soil and examined potential correlations between chemical and biochemical characteristics of solid fractions and their C and N turnover. The solid fractions were mixed with soil and incubated at 14°C for 120 days, during which CO2 evolution and inorganic N content of the solid fractions were determined. A two-parameter exponential function fitted to the individual solid fraction C mineralisation patterns explained 98% of the data variation, while a three-parameter Monod-type equation fitted to the net N mineralisation patterns explained 89% of the variation. Between 5% and 45% of initially added C was mineralised within the incubation period, with the largest proportion tending to be mineralised from simple mechanically separated solid fractions (MEC). Nitrogen was initially immobilised by the majority of solid fractions. Solid fractions from decanter centrifuged, anaerobically digested slurry (DEC) and chemically pre-treated and separated slurry (KEM) began to re-mineralise N after 20–40 days, whereas N was continuously immobilised from MEC solid fractions. The carbon mineralisation rate constant was correlated with the C content in the neutral detergent soluble (NDS), hemicellulose and cellulose fractions and the N content in NDS. Net nitrogen mineralisation was correlated with the C/Norg ratio of solid fractions, the N content of NDS and the C content of hemicellulose and cellulose.

Recovery of Small Infectious PrPres Aggregates from Prion-infected Cultured Cells

Prion diseases are characterized by deposits of abnormal conformers of the PrP protein. Although large aggregates of proteinase K-resistant PrP (PrP(res)) are infectious, the precise relationships between aggregation state and infectivity remain to be established. In this study, we have fractionated detergent lysates from prion-infected cultured cells by differential ultracentrifugation and ultrafiltration and have characterized a previously unnoticed PrP species. This abnormal form is resistant to proteinase K digestion but, in contrast to typical aggregated PrP(res), remains in the soluble fraction at intermediate centrifugal forces and is not retained by filters of 300-kDa cutoff. Cell-based assay and inoculation to animals demonstrate that these entities are infectious. The finding that cell-derived small infectious PrP(res) aggregates can be recovered in the absence of strong in vitro denaturating treatments now gives a biological basis for investigating the role of small PrP aggregates in the pathogenicity and/or the multiplication cycle of prions.

TcNDPK2, a Trypanosoma cruzi microtubule-associated nucleoside diphosphate kinase

Nucleoside diphosphate kinases (NDPKs) are enzymes required to preserve the intracellular nucleoside phosphate equilibrium. Trypanosoma cruzi has four putative nucleoside diphosphate kinases with unidentified biological roles and subcellular localization. TcNDPK2 has an N-terminal domain (DM10) with unknown function, which defines a subgroup of NDPKs distributed in a wide variety of organisms. Digitonin extraction demonstrated that this isoform is distributed in detergent soluble and insoluble fractions. Fluorescence microscopy showed that TcNDPK2 alone or fused to GFP was localized in cytoskeleton and flagella. TcNDPK2 was also detected by Western blot in purified polymerized tubulin and flagellar samples. In parasites expressing DM10 fused with GFP, the fluorescence was localized in cytoskeleton and flagellum with an identical pattern to TcNDPK2. This constitutes the first report that could give insights on the role of DM10 domains in NDPKs and also the identification of the first T. cruzi peptide that contains a microtubule association domain.

Kinetics fermentation and gas production of the neutral detergent-soluble fraction of fresh forage, silage and hay of Avena sativa

Neutral detergent treatment to evaluate the structural polysaccharides (NDF) removes soluble sugars, pectin substances, starch and the non-carbohydrate fraction (Van Soest, 1991). Despite the widely recognized importance of neutral detergent-soluble carbohydrates (NDS) in the early stages of forage digestion, little is known about their digestion kinetics because most in vitro methods have studied the disappearance of insoluble cell wall components. However, knowledge of the degradation characteristics of both fractions is very important to formulate balanced diets for high-yielding animals. Using the in vitro gas production system........

Differential subcellular distribution of rat brain dopamine receptors and subtype-specific redistribution induced by cocaine

We investigated the subcellular distribution of dopamine D 1, D 2 and D 5 receptor subtypes in rat frontal cortex, and examined whether psychostimulant-induced elevation of synaptic dopamine could alter the receptor distribution. Differential detergent solubilization and density gradient centrifugation were used to separate various subcellular fractions, followed by semi-quantitative determination of the relative abundance of specific receptor proteins in each fraction. D 1 receptors were predominantly localized to detergent-resistant membranes, and a portion of these receptors also floated on sucrose gradients. These properties are characteristic of proteins found in lipid rafts and caveolae. D 2 receptors exhibited variable distribution between cytoplasmic, detergent-soluble and detergent-resistant membrane fractions, yet were not present in buoyant membranes. Most D 5 receptor immunoreactivity was distributed into the cytoplasmic fraction, failing to sediment at forces up to 300,000 g, while the remainder was localized to detergent-soluble membranes in cortex. D 5 receptors were undetectable in detergent-resistant fractions or raft-like subdomains. Following daily cocaine administration for seven days, a significant portion of D 1 receptors translocated from detergent-resistant membranes to detergent-soluble membranes and the cytoplasmic fraction. The distributions of D 5 and D 2 receptor subtypes were not significantly altered by cocaine treatment. These data imply that D 5 receptors are predominantly cytoplasmic, D 2 receptors are diffusely distributed within the cell, whereas D 1 receptors are mostly localized to lipid rafts within the rat frontal cortex. Dopamine receptor subtype localization is susceptible to modulation by pharmacological manipulations that elevate synaptic dopamine, however the functional implications of such drug-induced receptor warrant further investigation.