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Visualization of the cocaine-sensitive dopamine transporter with ligand-conjugated quantum dots.
Kovtun O, Tomlinson ID, Sakrikar DS, Chang JC, Blakely RD, Rosenthal SJ
(2011) ACS Chem Neurosci 2: 370-8
MeSH Terms: Animals, Cell Adhesion, Cells, Cocaine, Dopamine Plasma Membrane Transport Proteins, Dopamine Uptake Inhibitors, Flow Cytometry, HeLa Cells, Humans, Indicators and Reagents, Ligands, Microscopy, Confocal, Phorbol Esters, Protein Kinase C, Quantum Dots, Streptavidin
Show Abstract · Added December 5, 2013
The presynaptic dopamine (DA) transporter is responsible for DA inactivation following release and is a major target for the psychostimulants cocaine and amphetamine. Dysfunction and/or polymorphisms in human DAT (SLC6A3) have been associated with schizophrenia, bipolar disorder, Parkinson's disease, and attention-deficit hyperactivity disorder (ADHD). Despite the clinical importance of DAT, many uncertainties remain regarding the transporter's regulation, in part due to the poor spatiotemporal resolution of conventional methodologies and the relative lack of efficient DAT-specific fluorescent probes. We developed a quantum dot-based labeling approach that uses a DAT-specific, biotinylated ligand, 2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane (IDT444), that can be bound by streptavidin-conjugated quantum dots. Flow cytometry and confocal microscopy were used to detect DAT in stably and transiently transfected mammalian cells. IDT444 is useful for quantum-dot-based fluorescent assays to monitor DAT expression, function, and plasma membrane trafficking in living cells as evidenced by the visualization of acute, protein-kinase-C (PKC)-dependent DAT internalization.
2 Communities
3 Members
0 Resources
16 MeSH Terms
A flow cytometry-based dopamine transporter binding assay using antagonist-conjugated quantum dots.
Kovtun O, Ross EJ, Tomlinson ID, Rosenthal SJ
(2012) Chem Commun (Camb) 48: 5428-30
MeSH Terms: Binding, Competitive, Biotin, Dopamine, Dopamine Antagonists, Dopamine Plasma Membrane Transport Proteins, Flow Cytometry, Fluorescent Dyes, Gene Expression Regulation, HEK293 Cells, High-Throughput Screening Assays, Humans, Phorbol Esters, Piperazines, Polyethylene Glycols, Quantum Dots, Spectrometry, Fluorescence
Show Abstract · Added May 27, 2014
Here we present the development and validation of a flow cytometry-based dopamine transporter (DAT) binding assay that uses antagonist-conjugated quantum dots (QDs). We anticipate that our QD-based assay is of immediate value to the high throughput screening of novel DAT modulators.
0 Communities
2 Members
0 Resources
16 MeSH Terms
PLD1 rather than PLD2 regulates phorbol-ester-, adhesion-dependent and Fc{gamma}-receptor-stimulated ROS production in neutrophils.
Norton LJ, Zhang Q, Saqib KM, Schrewe H, Macura K, Anderson KE, Lindsley CW, Brown HA, Rudge SA, Wakelam MJ
(2011) J Cell Sci 124: 1973-83
MeSH Terms: Animals, Cell Adhesion, Mice, Mice, Knockout, Neutrophils, Phorbol Esters, Phospholipase D, Reactive Oxygen Species, Receptors, IgG, Signal Transduction, Tetradecanoylphorbol Acetate
Show Abstract · Added March 21, 2013
The signalling lipid phosphatidic acid (PA) is generated by the hydrolysis of phosphatidylcholine (PC), which is catalysed by phospholipase D (PLD) enzymes. Neutrophils, important cells of the innate immune system, maintain the body's defence against infection. Previous studies have implicated PLD-generated PA in neutrophil function; these have relied heavily on the use of primary alcohols to act as inhibitors of PA production. The recent development of isoform-selective small molecule inhibitors and the generation of a knockout mouse model provide us with accurate tools to study the role of PLDs in neutrophil responses. We show that PLD1 is a regulator of phorbol-ester-, chemoattractant, adhesion-dependent and Fcγ-receptor-stimulated production of reactive oxygen species (ROS) in neutrophils. Significantly we found that this role of PLD is isoform specific: the absence of PLD2 does not negatively affect these processes. Contrary to expectation, other functions required for an efficient immune response operate effectively in Pld2-deficient neutrophils or when both isoforms are inhibited pharmacologically. We conclude that although PLD1 does have important regulatory roles in neutrophils, the field has been confused by the use of primary alcohols; now that gold standard Pld-knockout mouse models are available, previous work might need to be reassessed.
0 Communities
1 Members
0 Resources
11 MeSH Terms
Calcium-dependent interactions of the human norepinephrine transporter with syntaxin 1A.
Sung U, Blakely RD
(2007) Mol Cell Neurosci 34: 251-60
MeSH Terms: Animals, Calcium, Cells, Cultured, Cerebral Cortex, Cricetinae, Cricetulus, Drug Interactions, Enzyme Activation, Humans, Models, Biological, Neurons, Norepinephrine Plasma Membrane Transport Proteins, Phorbol Esters, Protein Kinase C, Protein Transport, Synaptosomes, Syntaxin 1, Time Factors, Transfection
Show Abstract · Added July 10, 2013
The norepinephrine (NE) transporter (NET) terminates noradrenergic signaling by clearing released NE at synapses. The activity of NET can be rapidly regulated by depolarization and receptor activation via Ca2+ and kinase/phosphatase-linked pathways. The SNARE protein syntaxin 1A (SYN1A) interacts with NET and influences transporter surface trafficking and catalytic activity. In this study, we establish a link between changes in intracellular Ca2+ and SYN1A/NET interactions. SYN1A influenced NE transport only in the presence of Ca2+ in brain cortical synaptosomes. Although NET/SYN1A associations were sensitive to manipulations of Ca2+ in CHO cells, in vitro binding experiments using purified NET and SYN1A fusion proteins demonstrated a lack of direct Ca2+ sensitivity. Disruption of NET/SYN1A interaction abolished inhibition of NE transport by phorbol ester (PMA) to activate protein kinase C (PKC), but had no effect on transport inhibition by the Ca2+ calmodulin kinase (CaMK) inhibitor KN93. Furthermore, PMA enhanced Ca2+-dependent modulation of NE transport in synaptosomes. Our data reveal roles for SYN1A in the Ca2+-dependent regulation of NET, likely reliant on regulation by PKC signaling, but independent of CaMK.
1 Communities
1 Members
0 Resources
19 MeSH Terms
Expression studies of naturally occurring human dopamine transporter variants identifies a novel state of transporter inactivation associated with Val382Ala.
Mazei-Robison MS, Blakely RD
(2005) Neuropharmacology 49: 737-49
MeSH Terms: Alanine, Amphetamine, Animals, Biotinylation, COS Cells, Cattle, Cell Line, Chlorocebus aethiops, Cocaine, Dopamine, Dopamine Plasma Membrane Transport Proteins, Dopamine Uptake Inhibitors, Drug Interactions, Electrophoresis, Polyacrylamide Gel, Gene Expression, Gene Expression Regulation, Genetic Variation, Humans, Mice, Models, Biological, Mutagenesis, Neuroblastoma, Norepinephrine, Phorbol Esters, Protein Transport, Rats, Time Factors, Transfection, Valine
Show Abstract · Added July 10, 2013
Multiple, rare, human dopamine (DA) transporter (hDAT, SLC6A3) coding variants have been described, though to date they have been incompletely characterized. Here we present studies analyzing the function and regulation of five naturally occurring coding variants, V55A, R237Q, V382A, A559V and E602G, expressed in COS-7 and SH-SY5Y cells. All variants, except V382A, exhibited levels of surface protein expression and DA transport activity comparable to hDAT. V382A, divergent at the most highly conserved residue among reported hDAT variants, exhibited significantly diminished surface expression, likely derived from inefficient plasma membrane delivery. Moreover, a greater expression of V382A protein was required to achieve comparable levels of transport to hDAT, consistent with a loss of transport function. V382A displayed a decrease in sensitivity to phorbol ester (PMA)-induced internalization, as well as an altered substrate selectivity for DA versus norepinephrine (NE). Analysis of PMA-induced V382A internalization revealed a trafficking-independent action of PMA, consistent with the existence of a surface-localized, transport-inactive state.
1 Communities
1 Members
0 Resources
29 MeSH Terms
Bves modulates epithelial integrity through an interaction at the tight junction.
Osler ME, Chang MS, Bader DM
(2005) J Cell Sci 118: 4667-78
MeSH Terms: Animals, Avian Proteins, Biological Transport, Cadherins, Calcium, Cell Adhesion, Cell Adhesion Molecules, Cell Line, Cell Membrane, Chickens, Dogs, Epithelial Cells, Golgi Apparatus, Humans, Membrane Proteins, Muscle Proteins, Mutagenesis, Occludin, Phorbol Esters, Phosphoproteins, Protein Binding, Tight Junctions, Zonula Occludens-1 Protein
Show Abstract · Added September 28, 2015
We first identified Bves (blood vessel/epicardial substance) as a transmembrane protein that localized to the lateral compartment of the epithelial epicardium. Bves traffics to sites of cell-cell contact in cultured epicardial cells and promotes adhesion following transfection into non-adherent fibroblastic L-cells, reminiscent of a cell adhesion molecule. Currently, no function for Bves in relation to epithelial cell adhesion has been identified. We hypothesize that Bves plays a role at cell junctions to establish and/or modulate cell adhesion or cell-cell interactions in epithelial cell types. In this study, we demonstrate that Bves regulates epithelial integrity and that this function may be associated with a role at the tight junction (TJ). We report that Bves localizes with ZO-1 and occludin, markers of the TJ, in polarized epithelial cell lines and in vivo. We find that the behavior of Bves following low Ca2+ challenge or TPA treatment mimics that observed for ZO-1 and is distinct from adherens junction proteins such as E-cadherin. Furthermore, GST pull-down experiments show an interaction between ZO-1 and the intracellular C-terminal tail of Bves. Finally, we demonstrate that Bves modulates tight junction integrity, as indicated by the loss of transepithelial resistance and junction protein localization at the membrane following Bves knock-down in cultured cells. This study is the first to identify a function for Bves in epithelia and supports the hypothesis that Bves contributes to establishment and/or maintenance of epithelial cell integrity.
1 Communities
1 Members
0 Resources
23 MeSH Terms
Accessory elements, flanking DNA sequence, and promoter context play key roles in determining the efficacy of insulin and phorbol ester signaling through the malic enzyme and collagenase-1 AP-1 motifs.
Ayala JE, Streeper RS, Svitek CA, Goldman JK, Oeser JK, O'Brien RM
(2002) J Biol Chem 277: 27935-44
MeSH Terms: Base Sequence, Cell Line, Chloramphenicol O-Acetyltransferase, Collagenases, DNA Primers, Gene Expression Regulation, Enzymologic, HeLa Cells, Humans, Insulin, Malate Dehydrogenase, Mutagenesis, Oligodeoxyribonucleotides, Phorbol Esters, Polymerase Chain Reaction, Promoter Regions, Genetic, Recombinant Proteins, Transcription Factor AP-1, Transcription Factors, Transfection, beta-Galactosidase
Show Abstract · Added July 10, 2013
Insulin stimulates malic enzyme (ME)-chloramphenicol acetyltransferase (CAT) and collagenase-1-CAT fusion gene expression in H4IIE cells through identical activator protein-1 (AP-1) motifs. In contrast, insulin and phorbol esters only stimulate collagenase-1-CAT and not ME-CAT fusion gene expression in HeLa cells. The experiments in this article were designed to explore the molecular basis for this differential cell type- and gene-specific regulation. The results highlight the influence of three variables, namely promoter context, AP-1 flanking sequence, and accessory elements that modulate insulin and phorbol ester signaling through the AP-1 motif. Thus, fusion gene transfection and proteolytic clipping gel retardation assays suggest that the AP-1 flanking sequence affects the conformation of AP-1 binding to the collagenase-1 and ME AP-1 motifs such that it selectively binds the latter in a fully activated state. However, this influence of ME AP-1 flanking sequence is dependent on promoter context. Thus, the ME AP-1 motif will mediate both an insulin and phorbol ester response in HeLa cells when introduced into either the collagenase-1 promoter or a specific heterologous promoter. But even in the context of the collagenase-1 promoter, the effects of both insulin and phorbol esters, mediated through the ME AP-1 motif are dependent on accessory factors.
0 Communities
1 Members
0 Resources
20 MeSH Terms
Cocaine and antidepressant-sensitive biogenic amine transporters exist in regulated complexes with protein phosphatase 2A.
Bauman AL, Apparsundaram S, Ramamoorthy S, Wadzinski BE, Vaughan RA, Blakely RD
(2000) J Neurosci 20: 7571-8
MeSH Terms: Animals, Antidepressive Agents, Biogenic Amines, Biological Transport, Carrier Proteins, Cell Line, Cocaine, Dopamine Plasma Membrane Transport Proteins, Humans, Macromolecular Substances, Marine Toxins, Membrane Glycoproteins, Membrane Transport Proteins, Nerve Tissue Proteins, Norepinephrine Plasma Membrane Transport Proteins, Okadaic Acid, Oxazoles, Phorbol Esters, Phosphoprotein Phosphatases, Phosphorylation, Precipitin Tests, Protein Kinase C, Protein Phosphatase 1, Protein Phosphatase 2, Protein Transport, Serotonin, Serotonin Plasma Membrane Transport Proteins, Symporters, Transfection
Show Abstract · Added December 10, 2013
Presynaptic transporter proteins regulate the clearance of extracellular biogenic amines after release and are important targets for multiple psychoactive agents, including amphetamines, cocaine, and antidepressant drugs. Recent studies reveal that dopamine (DA), norepinephrine (NE), and serotonin (5-HT) transporters (DAT, NET, and SERT, respectively) are rapidly regulated by direct or receptor-mediated activation of cellular kinases, particularly protein kinase C (PKC). With SERTs, PKC activation results in activity-dependent transporter phosphorylation and sequestration. Protein phosphatase 1/2A (PP1/PP2A) inhibitors, such as okadaic acid (OA) and calyculin A, also promote SERT phosphorylation and functional downregulation. How kinase, phosphatase, and transporter activities are linked mechanistically is unclear. In the present study, we found that okadaic acid-sensitive phosphatase activity is enriched in SERT immunoprecipitates from human SERT stably transfected cells. Moreover, blots of these immunoprecipitates reveal the presence of PP2A catalytic subunit (PP2Ac), findings replicated using brain preparations. Whole-cell treatments with okadaic acid or calyculin A diminished SERT/PP2Ac associations. Phorbol esters, which trigger SERT phosphorylation, also diminish SERT/PP2Ac associations, effects that can be blocked by PKC antagonists as well as the SERT substrate 5-HT. Similar transporter/PP2Ac complexes were also observed in coimmunoprecipitation studies with NETs and DATs. Our findings provide evidence for the existence of regulated heteromeric assemblies involving biogenic amine transporters and PP2A and suggest that the dynamic stability of these complexes may govern transporter phosphorylation and sequestration.
1 Communities
2 Members
0 Resources
29 MeSH Terms
Angiotensin II attenuates renal cortical cyclooxygenase-2 expression.
Cheng HF, Wang JL, Zhang MZ, Miyazaki Y, Ichikawa I, McKanna JA, Harris RC
(1999) J Clin Invest 103: 953-61
MeSH Terms: Angiotensin II, Angiotensin Receptor Antagonists, Angiotensin-Converting Enzyme Inhibitors, Animals, Captopril, Cyclooxygenase 2, Cyclooxygenase 2 Inhibitors, Cyclooxygenase Inhibitors, Gene Expression Regulation, Enzymologic, Isoenzymes, Kidney Cortex, Losartan, Male, Mice, Mice, Knockout, Phorbol Esters, Prostaglandin-Endoperoxide Synthases, RNA, Messenger, Rabbits, Rats, Rats, Sprague-Dawley, Receptor, Angiotensin, Type 1, Receptor, Angiotensin, Type 2, Receptors, Angiotensin, Renin, Renin-Angiotensin System
Show Abstract · Added January 28, 2014
We have previously shown that in rat renal cortex, cyclooxygenase-2 (COX-2) expression is localized to cTALH cells in the region of the macula densa, and that dietary salt restriction increases COX-2 expression. Administration of the angiotensin converting inhibitor, captopril, further increased COX-2 mRNA and renal cortical COX-2 immunoreactivity, with the most pronounced expression in the macula densa. Administration of an AT1 receptor antagonist, losartan, also significantly increased cortical COX-2 mRNA expression and COX-2 immunoreactivity. Mutant mice homozygous for both Agtr1a and Agtr1b null mutations (Agtr1a-/-,Agtr1b-/-) demonstrated large increases in immunoreactive COX-2 expression inthe cTALH/macula densa. To determine whether increased COX-2expression in response to ACE inhibition mediated increases in renin production, rats were treated with captopril for one week with or without the specific COX-2 inhibitor, SC58236. Plasma renin activity increased significantly in the captropril group, and this increase was significantly inhibited by simultaneous treatment with SC58236. Thus, these studies indicated that angiotensin II inhibitors augment upregulation of renal cortical COX-2 in states of volume depletion, suggesting that negative feedback by the renin-angiotensin system modulates renal cortical COX-2 expression and that COX-2 is a mediator of increased renin production in response to inhibition of angiotension II production.
1 Communities
2 Members
0 Resources
26 MeSH Terms
Regulation of CYP11A gene expression in bovine ovarian granulosa cells in primary culture by cAMP and phorbol esters is conferred by a common cis-acting element.
Lauber ME, Picton HM, Begeot M, Momoi K, Waterman MR, Simpson ER
(1993) Mol Cell Endocrinol 94: 235-42
MeSH Terms: Animals, Base Sequence, Cattle, Cells, Cultured, Cholesterol Side-Chain Cleavage Enzyme, Colforsin, Cyclic AMP, Cyclic AMP-Dependent Protein Kinases, Cytochrome P-450 Enzyme System, DNA, Female, Gene Expression Regulation, Enzymologic, Genes, Reporter, Granulosa Cells, Molecular Sequence Data, Phorbol Esters, Protein Kinase C, RNA, Messenger, Second Messenger Systems, Steroid 17-alpha-Hydroxylase, Tetradecanoylphorbol Acetate, Transfection
Show Abstract · Added February 12, 2015
Production and secretion of steroid hormones throughout the ovarian cycle occurs in a highly episodic and coordinated fashion that requires precise and finely tuned regulatory mechanisms. The regulation of ovarian steroidogenesis by the gonadotropin follicle stimulating hormone (FSH) and luteinizing hormone (LH) as well as by other factors occurs, at least in part, at the level of expression of the genes encoding steroidogenic enzymes. The present study is aimed at the elucidation of regulatory mechanisms by which cyclic adenosine monophosphate (cAMP) and protein kinase C regulate cytochrome P450scc (CYP11A) gene expression in bovine granulosa cells in primary culture. As a first step we characterized the bovine granulosa cell cultures with regard to regulation of P450scc activity and mRNA levels upon treatment with forskolin and/or the phorbol ester TPA. Forskolin, a potent stimulator of cAMP generation, increased both progesterone secretion and P450scc mRNA levels. In contrast, treatment with TPA alone decreased both basal progesterone production and P450scc mRNA accumulation. Co-treatment with forskolin and TPA decreased progesterone and P450scc mRNA levels as compared to forskolin treatment alone. The possibility that TPA interfered with the forskolin-stimulated cAMP production could be excluded because simultaneous treatment of granulosa cells with TPA and forskolin potentiated the formation of cAMP. In order to identify regulatory sequences within the 5' flanking region of the bovine CYP11A gene, chimeric DNA constructs comprizing regions of the CYP11A gene fused to a beta-globin-derived reporter gene were transfected into granulosa cells in primary culture.(ABSTRACT TRUNCATED AT 250 WORDS)
0 Communities
1 Members
0 Resources
22 MeSH Terms