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Analysis of the Phosphoinositide Composition of Subcellular Membrane Fractions.
Sarkes DA, Rameh LE
(2016) Methods Mol Biol 1376: 213-27
MeSH Terms: Cell Fractionation, Cell Membrane, Chromatography, High Pressure Liquid, Phosphatidylinositols, Staining and Labeling, Subcellular Fractions, Tritium
Show Abstract · Added November 26, 2018
Phosphoinositides play critical roles in the transduction of extracellular signals through the plasma membrane and also in endomembrane events important for vesicle trafficking and organelle function (Di Paolo and De Camilli, Nature 443(7112):651-657, 2006). The response triggered by these lipids is heavily dependent on the microenvironment in which they are found. HPLC analysis of labeled phosphoinositides allows quantification of the levels of each phosphoinositide species relative to their precursor, phosphatidylinositol. When combined with subcellular fractionation techniques, this strategy allows measurement of the relative phosphoinositide composition of each membrane fraction or organelle and determination of the microenvironment in which each species is enriched. Here, we describe the steps to separate and quantify total or localized phosphoinositides from cultured cells.
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MeSH Terms
Kinetic analysis of lauric acid hydroxylation by human cytochrome P450 4A11.
Kim D, Cha GS, Nagy LD, Yun CH, Guengerich FP
(2014) Biochemistry 53: 6161-72
MeSH Terms: Algorithms, Binding, Competitive, Biocatalysis, Cytochrome P-450 CYP4A, Cytochrome P-450 Enzyme System, Cytochromes b5, Deuterium, Electron Transport, Ferric Compounds, Ferrous Compounds, Humans, Hydroxylation, Kinetics, Lauric Acids, Models, Chemical, Models, Molecular, Oxidation-Reduction, Protein Binding, Protein Structure, Tertiary, Substrate Specificity, Tritium
Show Abstract · Added January 20, 2015
Cytochrome P450 (P450) 4A11 is the only functionally active subfamily 4A P450 in humans. P450 4A11 catalyzes mainly ω-hydroxylation of fatty acids in liver and kidney; this process is not a major degradative pathway, but at least one product, 20-hydroxyeicosatetraenoic acid, has important signaling properties. We studied catalysis by P450 4A11 and the issue of rate-limiting steps using lauric acid ω-hydroxylation, a prototypic substrate for this enzyme. Some individual reaction steps were studied using pre-steady-state kinetic approaches. Substrate and product binding and release were much faster than overall rates of catalysis. Reduction of ferric P450 4A11 (to ferrous) was rapid and not rate-limiting. Deuterium kinetic isotope effect (KIE) experiments yielded low but reproducible values (1.2-2) for 12-hydroxylation with 12-(2)H-substituted lauric acid. However, considerable "metabolic switching" to 11-hydroxylation was observed with [12-(2)H3]lauric acid. Analysis of switching results [Jones, J. P., et al. (1986) J. Am. Chem. Soc. 108, 7074-7078] and the use of tritium KIE analysis with [12-(3)H]lauric acid [Northrop, D. B. (1987) Methods Enzymol. 87, 607-625] both indicated a high intrinsic KIE (>10). Cytochrome b5 (b5) stimulated steady-state lauric acid ω-hydroxylation ∼2-fold; the apoprotein was ineffective, indicating that electron transfer is involved in the b5 enhancement. The rate of b5 reoxidation was increased in the presence of ferrous P450 mixed with O2. Collectively, the results indicate that both the transfer of an electron to the ferrous·O2 complex and C-H bond-breaking limit the rate of P450 4A11 ω-oxidation.
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21 MeSH Terms
Monitoring cholinergic activity during attentional performance in mice heterozygous for the choline transporter: a model of cholinergic capacity limits.
Paolone G, Mallory CS, Cherian AK, Miller TR, Blakely RD, Sarter M
(2013) Neuropharmacology 75: 274-85
MeSH Terms: Acetylcholine, Alkaloids, Animals, Atropine, Attention, Azocines, Cholinergic Antagonists, Conditioning, Operant, Female, Humans, Male, Mecamylamine, Membrane Transport Proteins, Mice, Mice, Inbred C57BL, Mice, Knockout, Protein Binding, Quinolizines, Sodium Channel Blockers, Tetrodotoxin, Tritium
Show Abstract · Added September 28, 2015
Reductions in the capacity of the human choline transporter (SLC5A7, CHT) have been hypothesized to diminish cortical cholinergic neurotransmission, leading to risk for cognitive and mood disorders. To determine the acetylcholine (ACh) release capacity of cortical cholinergic projections in a mouse model of cholinergic hypofunction, the CHT+/- mouse, we assessed extracellular ACh levels while mice performed an operant sustained attention task (SAT). We found that whereas SAT-performance-associated increases in extracellular ACh levels of CHT+/- mice were significantly attenuated relative to wildtype littermates, performance on the SAT was normal. Tetrodotoxin-induced blockade of neuronal excitability reduced both dialysate ACh levels and SAT performance similarly in both genotypes. Likewise, lesions of cholinergic neurons abolished SAT performance in both genotypes. However, cholinergic activation remained more vulnerable to the reverse-dialyzed muscarinic antagonist atropine in CHT+/- mice. Additionally, CHT+/- mice displayed greater SAT-disrupting effects of reverse dialysis of the nAChR antagonist mecamylamine. Receptor binding assays revealed a higher density of α4β2* nAChRs in the cortex of CHT+/- mice compared to controls. These findings reveal compensatory mechanisms that, in the context of moderate cognitive challenges, can overcome the performance deficits expected from the significantly reduced ACh capacity of CHT+/- cholinergic terminals. Further analyses of molecular and functional compensations in the CHT+/- model may provide insights into both risk and resiliency factors involved in cognitive and mood disorders.
Copyright © 2013 Elsevier Ltd. All rights reserved.
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21 MeSH Terms
Attention deficit/hyperactivity disorder-derived coding variation in the dopamine transporter disrupts microdomain targeting and trafficking regulation.
Sakrikar D, Mazei-Robison MS, Mergy MA, Richtand NW, Han Q, Hamilton PJ, Bowton E, Galli A, Veenstra-Vanderweele J, Gill M, Blakely RD
(2012) J Neurosci 32: 5385-97
MeSH Terms: Adolescent, Amphetamine, Analysis of Variance, Attention Deficit Disorder with Hyperactivity, Bacterial Proteins, Benzylamines, Biotinylation, Calcium, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Cell Line, Transformed, Child, Child, Preschool, Cholera Toxin, Cohort Studies, Dopamine, Dopamine Plasma Membrane Transport Proteins, Dopamine Uptake Inhibitors, Dose-Response Relationship, Drug, Electrochemistry, Female, Humans, Immunoprecipitation, Luminescent Proteins, Male, Membrane Microdomains, Membrane Proteins, Piperazines, Polymorphism, Single Nucleotide, Protein Kinase Inhibitors, Protein Transport, Sulfonamides, Transfection, Tritium
Show Abstract · Added December 5, 2013
Attention deficit/hyperactivity disorder (ADHD) is the most commonly diagnosed disorder of school-age children. Although genetic and brain-imaging studies suggest a contribution of altered dopamine (DA) signaling in ADHD, evidence of signaling perturbations contributing to risk is largely circumstantial. The presynaptic, cocaine- and amphetamine (AMPH)-sensitive DA transporter (DAT) constrains DA availability at presynaptic and postsynaptic receptors following vesicular release and is targeted by the most commonly prescribed ADHD therapeutics. Using polymorphism discovery approaches with an ADHD cohort, we identified a hDAT (human DAT) coding variant, R615C, located in the distal C terminus of the transporter, a region previously implicated in constitutive and regulated transporter trafficking. Here, we demonstrate that, whereas wild-type DAT proteins traffic in a highly regulated manner, DAT 615C proteins recycle constitutively and demonstrate insensitivity to the endocytic effects of AMPH and PKC (protein kinase C) activation. The disrupted regulation of DAT 615C parallels a redistribution of the transporter variant away from GM1 ganglioside- and flotillin1-enriched membranes, and is accompanied by altered CaMKII (calcium/calmodulin-dependent protein kinase II) and flotillin-1 interactions. Using C-terminal peptides derived from wild-type DAT and the R615C variant, we establish that the DAT 615C C terminus can act dominantly to preclude AMPH regulation of wild-type DAT. Mutagenesis of DAT C-terminal sequences suggests that phosphorylation of T613 may be important in sorting DAT between constitutive and regulated pathways. Together, our studies support a coupling of DAT microdomain localization with transporter regulation and provide evidence of perturbed DAT activity and DA signaling as a risk determinant for ADHD.
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33 MeSH Terms
Monomeric rhodopsin is sufficient for normal rhodopsin kinase (GRK1) phosphorylation and arrestin-1 binding.
Bayburt TH, Vishnivetskiy SA, McLean MA, Morizumi T, Huang CC, Tesmer JJ, Ernst OP, Sligar SG, Gurevich VV
(2011) J Biol Chem 286: 1420-8
MeSH Terms: Amino Acid Sequence, Animals, Arrestins, Cattle, Electrochemistry, Fluorescence, G-Protein-Coupled Receptor Kinase 1, Leucine, Lipids, Molecular Sequence Data, Mutation, Phosphorylation, Protein Binding, Rhodopsin, Signal Transduction, Tritium, Vision, Ocular, beta-Arrestins
Show Abstract · Added December 10, 2013
G-protein-coupled receptor (GPCR) oligomerization has been observed in a wide variety of experimental contexts, but the functional significance of this phenomenon at different stages of the life cycle of class A GPCRs remains to be elucidated. Rhodopsin (Rh), a prototypical class A GPCR of visual transduction, is also capable of forming dimers and higher order oligomers. The recent demonstration that Rh monomer is sufficient to activate its cognate G protein, transducin, prompted us to test whether the same monomeric state is sufficient for rhodopsin phosphorylation and arrestin-1 binding. Here we show that monomeric active rhodopsin is phosphorylated by rhodopsin kinase (GRK1) as efficiently as rhodopsin in the native disc membrane. Monomeric phosphorylated light-activated Rh (P-Rh*) in nanodiscs binds arrestin-1 essentially as well as P-Rh* in native disc membranes. We also measured the affinity of arrestin-1 for P-Rh* in nanodiscs using a fluorescence-based assay and found that arrestin-1 interacts with monomeric P-Rh* with low nanomolar affinity and 1:1 stoichiometry, as previously determined in native disc membranes. Thus, similar to transducin activation, rhodopsin phosphorylation by GRK1 and high affinity arrestin-1 binding only requires a rhodopsin monomer.
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18 MeSH Terms
Allosteric potentiators of metabotropic glutamate receptor subtype 1a differentially modulate independent signaling pathways in baby hamster kidney cells.
Sheffler DJ, Conn PJ
(2008) Neuropharmacology 55: 419-27
MeSH Terms: Allosteric Regulation, Amino Acids, Animals, Binding, Competitive, Calcium, Cell Line, Transformed, Cricetinae, Cyclic AMP, Dose-Response Relationship, Drug, Excitatory Amino Acid Agonists, Excitatory Amino Acid Antagonists, Glutamic Acid, Mitogen-Activated Protein Kinase 3, Pyrans, Quinolines, Radioligand Assay, Receptors, Metabotropic Glutamate, Signal Transduction, Time Factors, Transfection, Tritium, Xanthenes
Show Abstract · Added February 19, 2015
Recent studies suggest that subtype specific activators of metabotropic glutamate receptors (mGluRs) have exciting potential for the development of novel treatment strategies for numerous psychiatric and neurological disorders. A number of positive allosteric modulators (PAMs) have been identified that are highly selective for mGluR1, including the compounds Ro 01-6128, Ro 67-4853, and Ro 67-7476. These PAMs have been previously found to interact with a site distinct from that of negative allosteric modulators (NAMs), typified by R214127. These mGluR1 PAMs do not have an effect on baseline calcium levels but induce leftward shifts in the concentration-response of mGluR1 to agonists. However, their effects on a variety of signaling pathways and their mechanism of action have not been fully explored and are of critical importance for further development of mGluR1 allosteric modulators as novel drugs. In baby hamster kidney (BHK) cells, mGluR1 activates calcium mobilization, cAMP production, and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation; signaling cascades which are distinct and differentially regulated. In contrast to their effects on calcium mobilization, these compounds were found to activate ERK1/2 phosphorylation in the absence of exogenously added agonist, an effect that was fully blocked by both orthosteric (LY341495) and allosteric (R214127) mGluR1 antagonists. The mGluR1 PAMs were also found to activate cAMP production in the absence of agonist. Thus, these mGluR1 PAMs have qualitatively different effects on a variety of mGluR1-mediated signal transduction cascades. Together, these data provide further evidence that allosteric compounds can differentially modulate the coupling of a single receptor to independent signaling pathways or act in a system-dependent manner.
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22 MeSH Terms
Long chain fatty acid uptake in vivo: comparison of [125I]-BMIPP and [3H]-bromopalmitate.
Shearer J, Coenen KR, Pencek RR, Swift LL, Wasserman DH, Rottman JN
(2008) Lipids 43: 703-11
MeSH Terms: Animals, Bromine Compounds, Fatty Acids, Iodine Radioisotopes, Iodobenzenes, Male, Organ Specificity, Palmitates, Rats, Rats, Sprague-Dawley, Tritium
Show Abstract · Added December 10, 2013
Insulin resistance is characterized by increased metabolic uptake of fatty acids. Accordingly, techniques to examine in vivo shifts in fatty acid metabolism are of value in both clinical and experimental settings. Partially metabolizable long chain fatty acid (LCFA) tracers have been recently developed and employed for this purpose: [9,10-3H]-(R)-2-bromopalmitate ([3H]-BROMO) and [125I]-15-(rho-iodophenyl)-3-R,S-methylpentadecanoic acid ([125I]-BMIPP). These analogues are taken up like native fatty acids, but once inside the cell do not directly enter beta-oxidation. Rather, they become trapped in the slower processes of omega and alpha-oxidation. Study aims were to (1) simultaneously assess and compare [3H]-BROMO and [125I]-BMIPP and (2) determine if tracer breakdown is affected by elevated metabolic demands. Catheters were implanted in a carotid artery and jugular vein of Sprague-Dawley rats. Following 5 days recovery, fasted animals (5 h) underwent a rest (n = 8) or exercise (n = 8) (0.6 mi/h) protocol. An instantaneous bolus containing both [3H]-BROMO and [125I]-BMIPP was administered to determine LCFA uptake. No significant difference between [125I]-BMIPP and [3H]-BROMO uptake was found in cardiac or skeletal muscle during rest or exercise. In liver, rates of uptake were more than doubled with [3H]-BROMO compared to [125I]-BMIPP. Analysis of tracer conversion by TLC demonstrated no difference at rest. Exercise resulted in greater metabolism and excretion of tracers with approximately 37% and approximately 53% of [125I]-BMIPP and [3H]-BROMO present in conversion products at 40 min. In conclusion, [3H]-BROMO and [125I]-BMIPP are indistinguishable for the determination of tissue kinetics at rest in skeletal and cardiac muscle. Exercise preferentially exacerbates the breakdown of [3H]-BROMO, making [125I]-BMIPP the analogue of choice for prolonged (>30 min) experimental protocols with elevated metabolic demands.
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11 MeSH Terms
Prolonged glucocorticoid treatment decreases cannabinoid CB1 receptor density in the hippocampus.
Hill MN, Carrier EJ, Ho WS, Shi L, Patel S, Gorzalka BB, Hillard CJ
(2008) Hippocampus 18: 221-6
MeSH Terms: Animals, Anti-Inflammatory Agents, Arachidonic Acids, Corticosterone, Cyclohexanols, Dose-Response Relationship, Drug, Endocannabinoids, Glycerides, Hippocampus, Immunosuppressive Agents, Male, Polyunsaturated Alkamides, Rats, Rats, Long-Evans, Receptor, Cannabinoid, CB1, Tritium
Show Abstract · Added March 26, 2019
Experimental studies indicate a bidirectional, functional relationship between glucocorticoids and the endocannabinoid system; however, the effects of repeated glucocorticoid treatment on the endocannabinoid system have not been examined. In this study, we treated male rats with either a single dose or a 21-day course of treatment with corticosterone (20 mg/kg) and measured hippocampal cannabinoid CB(1) receptor expression and endocannabinoid content. The 21-day, but not the single, administration of corticosterone significantly reduced both the binding site density and amount of protein of the hippocampal cannabinoid CB(1) receptor without affecting affinity for the CB(1) receptor agonist, [(3)H]CP55940. With regard to hippocampal endocannabinoid content, acute corticosterone treatment resulted in a significant reduction in anandamide but did not affect 2-arachidonylglycerol, while repeated corticosterone treatment did not alter content of either anandamide or 2-arachidonylglycerol. These data support the hypothesis that the cannabinoid CB(1) receptor is under negative regulation by glucocorticoids in the hippocampus, and suggest that hippocampal cannabinoid CB(1) receptor signaling could be reduced under conditions associated with hypersecretion of glucocorticoids, such as chronic stress.
(c) 2007 Wiley-Liss, Inc.
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Juxtacrine activation of EGFR regulates claudin expression and increases transepithelial resistance.
Singh AB, Sugimoto K, Dhawan P, Harris RC
(2007) Am J Physiol Cell Physiol 293: C1660-8
MeSH Terms: Animals, Antibodies, Antigens, CD, Autocrine Communication, Calcium, Cell Line, Cell Membrane Permeability, Dogs, Electric Impedance, Epithelial Cells, ErbB Receptors, Heparin-binding EGF-like Growth Factor, Humans, Integrin beta1, Intercellular Signaling Peptides and Proteins, Inulin, Membrane Glycoproteins, Membrane Proteins, Phosphatidylinositol 3-Kinases, Phosphorylation, Protein Kinase Inhibitors, Proto-Oncogene Proteins c-akt, Rats, Sodium, Tetraspanin 29, Tight Junctions, Time Factors, Transfection, Tritium
Show Abstract · Added August 19, 2013
Heparin-binding (HB)-EGF, a ligand for EGF receptors, is synthesized as a membrane-anchored precursor that is potentially capable of juxtacrine activation of EGF receptors. However, the physiological importance of such juxtacrine signaling remains poorly described, due to frequent inability to distinguish effects mediated by membrane-anchored HB-EGF vs. mature "secreted HB-EGF." In our studies, using stable expression of a noncleavable, membrane-anchored rat HB-EGF isoform (MDCK(rat5aa) cells) in Madin-Darby canine kidney (MDCK) II cells, we observed a significant increase in transepithelial resistance (TER). Similar significant increases in TER were observed on stable expression of an analogous, noncleavable, membrane-anchored human HB-EGF construct (MDCK(human5aa) cells). The presence of noncleavable, membrane-anchored HB-EGF led to alterations in the expression of selected claudin family members, including a marked decrease in claudin-2 in MDCK(rat5aa) cells compared with the control MDCK cells. Reexpression of claudin-2 in MDCK(rat5aa) cells largely prevented the increases in TER. Ion substitution studies indicated decreased paracellular ionic permeability of Na(+) in MDCK(rat5aa) cells, further indicating that the altered claudin-2 expression mediated the increased TER seen in these cells. In a Ca(2+)-switch model, increased phosphorylation of EGF receptor and Akt was observed in MDCK(rat5aa) cells compared with the control MDCK cells, and inhibition of these pathways inhibited TER changes specifically in MDCK(rat5aa) cells. Therefore, we hypothesize that juxtacrine activation of EGFR by membrane-anchored HB-EGF may play an important role in the regulation of tight junction proteins and TER.
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29 MeSH Terms
Direct modulation of phospholipase D activity by Gbetagamma.
Preininger AM, Henage LG, Oldham WM, Yoon EJ, Hamm HE, Brown HA
(2006) Mol Pharmacol 70: 311-8
MeSH Terms: Binding Sites, Cell Line, Tumor, Choline, Dose-Response Relationship, Drug, Enzyme Activation, GTP-Binding Protein beta Subunits, GTP-Binding Protein gamma Subunits, Heterotrimeric GTP-Binding Proteins, Humans, Immunoblotting, Phospholipase D, Protein Binding, Transfection, Tritium
Show Abstract · Added March 19, 2013
Phospholipase D-mediated hydrolysis of phosphatidylcholine is stimulated by protein kinase C and the monomeric G proteins Arf, RhoA, Cdc42, and Rac1, resulting in complex regulation of this enzyme. Using purified proteins, we have identified a novel inhibitor of phospholipase D activity, Gbetagamma subunits of heterotrimeric G proteins. G protein-coupled receptor activation alters affinity between Galpha and Gbetagamma subunits, allowing subsequent interaction with distinct effectors. Gbeta1gamma1 inhibited phospholipase D1 and phospholipase D2 activity, and both Gbeta1gamma1 and Gbeta1gamma2 inhibited stimulated phospholipase D1 activity in a dosedependent manner in reconstitution assays. Reconstitution assays suggest this interaction occurs through the amino terminus of phospholipase D, because Gbeta1gamma1 is unable to inhibit an amino-terminally truncated phospholipase D construct, PLD1.d311, which like full-length phospholipase D isoforms, requires phosphatidylinositol-4,5-bisphosphate for activity. Furthermore, a truncated protein consisting of the amino-terminal region of phospholipase D containing the phox/pleckstrin homology domains was found to interact with Gbeta1gamma1, unlike the PLD1.d311 recombinant protein, which lacks this domain. In vivo, expressed recombinant Gbeta1gamma2 was also found to inhibit phospholipase D activity under basal and stimulated conditions in MDA-MB-231 cells, which natively express both phospholipase D1 and phospholipase D2. These data demonstrate that Gbetagamma directly regulates phospholipase D activity in vitro and suggest a novel mechanism to negatively regulate phospholipase D signaling in vivo.
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14 MeSH Terms