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1.
Vaccine ; 41(8): 1447-1456, 2023 02 17.
Article in English | MEDLINE | ID: mdl-36702691

ABSTRACT

Mucosal vaccines offer several advantages over transdermal vaccines, including the ability to acquire systemic and mucosal immunities. Smoking is a huge public health threat and major risk factor for various diseases that exacerbate or prolong respiratory symptoms and conditions. However, its impact on the efficacy of mucosal vaccines remains partially explored. Thus, this study investigates the effects of smoking on mucosal vaccine reactivity by assessing the induction of Th1 immunity, a vital response in infection defense. Cigarette smoke condensate was prepared as a substitute for mainstream smoke. We intranasally administered diphtheria toxoid as an antigen and natural CpG oligonucleotide G9.1, which enhances the Th1-type antibody (Ab) response in a plasmacytoid dendritic cells (pDCs) dependent manner, as an adjuvant to mice to assess the effect of cigarette smoke condensate on Ab responses. The mechanism of its effect was evaluated using human peripheral blood mononuclear cells and their pDC-rich fraction cultured with or without G9.1. In mice, cigarette smoke condensate tended to decrease diphtheria toxoid-specific Ab response, with a higher reduction in Th1-type IgG2 Ab response than in Th2-type IgG1 Ab response. In human peripheral blood mononuclear cells, cigarette smoke condensate significantly reduced the induction of IFN-α production by G9.1. Moreover, G9.1-induced increases in the CD83 expression in pDCs and the CD80 expression in DCs were suppressed via treatment with cigarette smoke condensate. Among the mechanisms suggested were decreased expression of toll-like receptor 9 mRNA, decreased expression of mRNA for IFN regulatory factor 7, and increased CpG methylation of its promoter region. The analysis of Tbet and GATA3 expressions revealed that cigarette smoke condensate exhibits Th1-directed immunostimulatory activity at a steady state but becomes more Th2-directed under G9.1 stimulation. In conclusion, smoking could reduce mucosal vaccine responses by decreasing pDC activation and, consequently, Th1-dominant immunity.


Subject(s)
Cigarette Smoking , Interferon-alpha , Animals , Humans , Mice , Dendritic Cells , Diphtheria Toxoid , Leukocytes, Mononuclear , RNA, Messenger/genetics , Smoking
2.
J Environ Radioact ; 138: 106-15, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25216467

ABSTRACT

Radioactive cesium ((134)Cs and (137)Cs) concentrations in invertebrates of benthic food web (10 taxonomic classes with 46 identified families) collected from wide areas off Fukushima Prefecture (3-500 m depth) were inspected from July 2011, four months after the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident, to August 2013 to elucidate time-series trends among taxa and areas. Cesium-137 was detected in seven classes (77% of 592 specimens). Higher (137)Cs concentrations within detected data were often found in areas near or south of the FDNPP, which is consistent with the reported spatial distribution of (137)Cs concentrations in highly contaminated seawater and sediments after the FDNPP accident. Overall (137)Cs concentrations in invertebrates, the maxima of which (290 Bq kg(-1)-wet in the sea urchin Glyptocidaris crenularis) were lower than in many demersal fishes, had decreased exponentially with time, and exhibited taxon-specific decreasing trends. Concentrations in Bivalvia and Gastropoda decreased clearly with respective ecological half-lives of 188 d and 102 d. In contrast, decreasing trends in Malacostraca and Polychaeta were more gradual, with longer respective ecological half-lives of 208 d and 487 d. Echinoidea showed no consistent trend, presumably because of effects of contaminated sediments taken into their digestive tract. Comparison of (137)Cs concentrations in the invertebrates and those in seawater and sediments suggest that contaminated sediments are the major source of continuing contamination in benthic invertebrates, especially in Malacostraca and Polychaeta.


Subject(s)
Cesium Radioisotopes/metabolism , Food Chain , Fukushima Nuclear Accident , Invertebrates/radiation effects , Water Pollutants, Radioactive/metabolism , Animals , Aquatic Organisms/radiation effects , Cesium Radioisotopes/analysis , Pacific Ocean , Radiation Monitoring , Seasons , Water Pollutants, Radioactive/analysis
3.
Front Pharmacol ; 2: 65, 2011.
Article in English | MEDLINE | ID: mdl-22025914

ABSTRACT

Nicotinic acetylcholine receptors (nAChRs) of the cerebral cortex and cerebellum of rats were evaluated by a radioligand binding assay, employing tissue segments, or homogenates as materials. [(3)H]-epibatidine specifically bound to nAChRs in rat cortex or cerebellum, but the dissociation constants for [(3)H]-epibatidine differed between segments and homogenates (187 pM for segments and 42 pM for homogenates in the cortex and 160 pM for segments and 84 pM for homogenates in the cerebellum). The abundance of total nAChRs was approximately 310 fmol/mg protein in the segments of cortex and 170 fmol/mg protein in the segments of cerebellum, which were significantly higher than those estimated in the homogenates (115 fmol/mg protein in the homogenates of the cortex and 76 fmol/mg protein in the homogenates of the cerebellum). Most of the [(3)H]-epibatidine binding sites in the cortex segments (approximately 70% of the population) showed high affinity for nicotine (pK(i) = 7.9), dihydro-ß-erythroidine, and cytisine, but the binding sites in the cerebellum segments had slightly lower affinity for nicotine (pK(i) = 7.1). An upregulation of nAChRs by chronic administration of nicotine was observed in the cortex segments but not in the cerebellum segments with [(3)H]-epibatidine as a ligand. The upregulation in the cortex was caused by a specific increase in the high-affinity sites for nicotine (probably α4ß2). The present study shows that the native environment of nAChRs is important for a precise quantitative as well as qualitative estimation of nAChRs in rat brain.

4.
J Neurochem ; 118(6): 958-67, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21740440

ABSTRACT

Signaling by muscarinic agonists is thought to result from the activation of cell surface acetylcholine receptors (mAChRs) that transmit extracellular signals to intracellular systems. In N1E-115 neuroblastoma cells, we detected both plasma membrane and intracellular M(1) -mAChRs using both biochemical and pharmacological methods. In intact cells, both plasma membrane and intracellular M(1) -mAChRs were detected by the hydrophobic ligand probe, 1-quinuclidinyl-[phenyl-4-(3) H]-benzilate ([(3) H]-QNB) whereas the hydrophilic probe, 1-[N-methyl-(3) H] scopolamine ([(3) H]-NMS), detected only cell surface receptors. These probes detected comparable numbers of receptors in isolated membrane preparations. Immunohistochemical studies with M(1) -mAChR antibody also detected both cell-surface and intracellular M(1) -mAChRs. Carbachol-stimulated phosphatidylinositol hydrolysis and Ca(2+) mobilization were completely inhibited by a cell-impermeable M(1) antagonist, muscarinic toxin -7 and the G(q/11) inhibitor YM-254890. However, carbachol-stimulated extracellular-regulated kinase 1/2 activation was unaffected by muscarinic toxin-7, but was blocked by the cell-permeable antagonist, pirenzepine. extracellular regulated kinase 1/2 phosphorylation was resistant to blockade of G(q/11) (YM-254890) and protein kinase C (bisindolylmaleimide I). Our data suggest that the geographically distinct M(1) -mAChRs (cell surface versus intracellular) can signal via unique signaling pathways that are differentially sensitive to cell-impermeable versus cell-permeable antagonists. Our data are of potential physiological relevance to signaling that affects both cognitive and neurodegenerative processes.


Subject(s)
Neuroblastoma/metabolism , Receptor, Muscarinic M1/metabolism , Animals , Atropine/pharmacology , Blotting, Western , Calcium/metabolism , Carbachol/metabolism , Carbachol/pharmacology , Cell Line, Tumor , Elapid Venoms/pharmacology , Enzyme Activation/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Immunohistochemistry , Inositol Phosphates/metabolism , Kinetics , Mice , Muscarinic Agonists/pharmacology , Muscarinic Antagonists/pharmacology , N-Methylscopolamine/pharmacology , Peptides, Cyclic/pharmacology , Pirenzepine/pharmacology , Protein Kinase Inhibitors/pharmacology , Quinuclidinyl Benzilate/pharmacology , Receptors, Cell Surface/drug effects
5.
J Pharmacol Exp Ther ; 339(1): 186-93, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21719469

ABSTRACT

Distinct pharmacological phenotypes of muscarinic acetylcholine receptors (mAChRs) have been proposed. We compared the pharmacological profiles of mAChRs in intact segments and homogenates of rat cerebral cortex and other tissues by using radioligand binding assays with [(3)H]N-methylscopolamine ([(3)H]NMS). Recombinant M(1) and M(3) mAChRs were also examined. The density of mAChRs detected by [(3)H]NMS binding to rat cerebral cortex segments and homogenates was the same (approximately 1400 fmol/mg tissue protein), but the dissociation constant of [(3)H]NMS was significantly different (1400-1700 pM in segments and 260 pM in homogenates). A wide variation in [(3)H]NMS binding affinity was also observed among the segments of other tissues (ranging from 139 pM in urinary bladder muscle to 1130 pM in the hippocampus). The mAChRs of cerebral cortex were composed of M(1), M(2), M(3), and M(4) subtypes, which showed typical subtype pharmacology in the homogenates. However, in the cortex segments the M(3) subtype showed a low selectivity for M(3) antagonists (darifenacin, solifenacin) and was not distinguished by the M(3) antagonists from the other subtypes. Recombinant M(1) and M(3) mAChRs showed high affinity for [(3)H]NMS and subtype-specific pharmacology for each tested ligand. The present binding study under conditions where tissue integrity was kept demonstrates a wide variation in [(3)H]NMS binding affinity among mAChRs of many rat tissues and the presence of an atypical M(3) phenotype in the cerebral cortex, suggesting that the pharmacological properties of mAChRs are not necessarily constant, rather they may be significantly modified by tissue integrity and tissue type.


Subject(s)
Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Receptors, Muscarinic/drug effects , Animals , Binding, Competitive/drug effects , CHO Cells , Cricetinae , Cricetulus , Data Interpretation, Statistical , In Vitro Techniques , Kinetics , Male , Muscarinic Antagonists/metabolism , Muscle, Smooth/metabolism , N-Methylscopolamine/metabolism , Phenotype , Rats , Rats, Wistar , Receptor, Muscarinic M2/metabolism , Receptor, Muscarinic M3/drug effects , Receptors, Muscarinic/metabolism
6.
Biochem Biophys Res Commun ; 409(4): 657-62, 2011 Jun 17.
Article in English | MEDLINE | ID: mdl-21616059

ABSTRACT

G-protein coupled receptor interacting scaffold protein (GISP) is a multi-domain, brain-specific protein derived from the A-kinase anchoring protein (AKAP)-9 gene. Using yeast two-hybrid screens to identify GISP interacting proteins we isolated the SUMO conjugating enzyme Ubc9. GISP interacts with Ubc9 in vitro, in heterologous cells and in neurons. SUMOylation is a post-translational modification in which the small protein SUMO is covalently conjugated to target proteins, modulating their function. Consistent with its interaction with Ubc9, we show that GISP is SUMOylated by both SUMO-1 and SUMO-2 in both in vitro SUMOylation assays and in mammalian cells. Intriguingly, SUMOylation of GISP in neurons occurs in an activity-dependent manner in response to chemical LTP. These data suggest that GISP is a novel neuronal SUMO substrate whose SUMOylation status is modulated by neuronal activity.


Subject(s)
Brain/metabolism , Nerve Tissue Proteins/metabolism , Sumoylation , A Kinase Anchor Proteins , Animals , Brain/cytology , COS Cells , Chlorocebus aethiops , Cytoskeletal Proteins , Nerve Tissue Proteins/genetics , Neurons/metabolism , Rats , SUMO-1 Protein/metabolism , Small Ubiquitin-Related Modifier Proteins/metabolism , Two-Hybrid System Techniques , Ubiquitin-Conjugating Enzymes/metabolism
7.
J Pharmacol Sci ; 113(2): 169-81, 2010.
Article in English | MEDLINE | ID: mdl-20508391

ABSTRACT

Two distinct alpha(1)-adrenoceptor phenotypes (alpha(1A)- and alpha(1L)-ARs) are known to originate from a single ADRA1A(alpha(1a)) gene by an as-yet-unknown mechanism. We hypothesized that an alpha(1a)-AR-interacting protein could generate the alpha(1L)-AR phenotype and we sought to identify such a protein and to examine its effects on the expression of alpha(1A) and alpha(1L) phenotypes. Cysteine-rich epidermal growth factor-like domain 1alpha (CRELD1alpha) was first identified using a yeast two-hybrid approach as an alpha(1a)-AR-interacting protein. Transfection of alpha(1a)-AR cDNA alone yielded Chinese hamster ovary (CHO) cells expressing alpha(1A)-ARs having a predominant high affinity site for prazosin, with a low proportion (<10%) of prazosin-low affinity sites (alpha(1L)-AR). Knockdown of endogenous CHO-CRELD1alpha [alpha(1a)-CKD(alpha(1A)-enhanced) cells] enhanced the expression of alpha(1A)-AR, whereas over-expression of CRELD1alpha reduced alpha(1A)-AR expression, yielding alpha(1a)-COE(alpha(1L)-dominant) cells expressing a high proportion (50%) of the alpha(1L)-AR phenotype. The ligand binding and functional agonist and antagonist profiles in alpha(1a)-CKD(alpha(1A)-enhanced) and alpha(1a)-COE(alpha(1L)-dominant) cell lines were entirely in accord with the alpha(1A)-AR and alpha(1L)-AR phenotypes observed in intact tissues. CRELD1alpha down-regulates expression of the alpha(1A)-AR, thereby enhancing the proportion of expression of the alpha(1L)-AR phenotype. The alpha(1L)-AR-expressing alpha(1a)-COE(alpha(1L)-dominant) cell line reflects accurately the phenotype of this AR observed in vivo and will facilitate development of alpha(1L)-AR-targeted drugs.


Subject(s)
CHO Cells , Cell Adhesion Molecules/isolation & purification , Cell Adhesion Molecules/physiology , Down-Regulation , Extracellular Matrix Proteins/isolation & purification , Extracellular Matrix Proteins/physiology , Receptors, Adrenergic, alpha-1/metabolism , Animals , Cell Adhesion Molecules/pharmacology , Cricetinae , Cricetulus , Extracellular Matrix Proteins/pharmacology , Female
8.
J Pharmacol Sci ; 112(4): 444-51, 2010.
Article in English | MEDLINE | ID: mdl-20424384

ABSTRACT

Muscarinic acetylcholine receptors (mAChRs) of rat cerebral cortex were evaluated using a tissue segment radioligand binding assay. [(3)H]-Quinuclidinyl benzilate (QNB, a hydrophobic ligand) specifically bound to mAChRs in the cortex segments. The total mAChRs level was approximately 2,000 fmol/mg protein, which was estimated after incubation for 120 min at 37 degrees C or for 8 h at 4 degrees C. These mAChRs were a mixture of high- and low-affinity sites for N-methylscopolamine (NMS) in a 70:30 ratio. In contrast, only a single high-affinity site for NMS was detected following incubation for 30 min at 37 degrees C, whose abundance was about 70% of that of the total mAChRs. Atropine showed a single affinity for mAChRs under all conditions. These indicate that mAChRs are constitutively expressed not only on plasma membrane sites but also at intracellular sites in rat cerebral cortex and that the receptors at both sites have different affinities for NMS. Acetylcholine completely inhibited [(3)H]-QNB binding to both mAChRs without any change in the subcellular distribution, suggesting the possibility that acetylcholine can access, and bind to, both mAChRs in intact tissue. Two different affinity states for acetylcholine were detected only in plasma membrane mAChRs at 37 degrees C. The present study demonstrates a unique subcellular distribution, and distinct pharmacological profiles, of mAChRs in rat cerebral cortex.


Subject(s)
Cerebral Cortex/metabolism , Receptors, Muscarinic/metabolism , Acetylcholine/pharmacology , Animals , Atropine/pharmacology , Cerebral Cortex/drug effects , In Vitro Techniques , Male , N-Methylscopolamine/pharmacology , Quinuclidinyl Benzilate/metabolism , Radioligand Assay , Rats , Rats, Wistar
9.
Int J Urol ; 17(1): 31-7, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19694838

ABSTRACT

Alpha(1)-adrenoceptors are involved in physiological functions such as urinary excretion and ejaculation in the lower urinary tract (LUT). Several alpha(1) antagonists are clinically used for the treatment of urinary obstruction in patients with benign prostatic hyperplasia. At present, three classical alpha(1)-adrenoceptor subtypes (alpha(1A), alpha(1B), and alpha(1D)) have been identified, among which the alpha(1A) and alpha(1D)-adrenoceptor subtypes have been regarded as the main targets of alpha(1) antagonist therapy for LUT symptoms. Prazosin has been used as a prototypic, classical antagonist, to characterize alpha(1)-adrenoceptors pharmacologically, (i.e. all classical alpha(1)-adrenoceptor subtypes show high-affinity for the drug). However, we found that alpha(1)-adrenoceptors in the LUT show atypical low-affinity for prazosin. Therefore, the concept alpha(1L)-receptor, which indicates alpha(1)-adrenoceptor(s) showing low-affinity for prazosin has been introduced. A recent study demonstrated that the alpha(1L)-adrenoceptor is a specific phenotype present in the many intact tissues including human LUT, and that it originates from the ADRA1A gene. Therefore, the alpha(1L)-adrenoceptor in the LUT is now re-defined as alpha(1A(L))-adrenoceptor. The physiological and pharmacological difference between classical alpha(1A(H),) and alpha(1A(L)) which is the native receptor expressed in the LUT is of special interest as it provides fundamental bases for urological alpha(1A)-adrenoceptor blocking pharmacotherapy. Here, we briefly review the alpha(1)-adrenoceptors in the LUT with special reference to phenotype-based (pharmacome) analysis.


Subject(s)
Receptors, Adrenergic, alpha-1 , Animals , Humans , Mice , Receptors, Adrenergic, alpha-1/genetics , Receptors, Adrenergic, alpha-1/physiology , Urethra/physiology , Urinary Bladder/physiology
10.
J Urol ; 183(2): 812-9, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20034639

ABSTRACT

PURPOSE: Although alpha(1L)-adrenoceptor is recognized as a target of alpha(1) antagonist therapy for benign prostatic hyperplasia, the most common techniques, such as immunohistochemistry and in situ hybridization, are not applicable to examine alpha(1L)-AR vs alpha(1A)-AR tissue distribution because alpha(1L)-AR is now considered another phenotype sharing the alpha(1A)-AR gene and protein molecule. We labeled the alpha(1A) and alpha(1L)-adrenoceptor selective antagonist silodosin (Kissei Pharmaceutical, Matsumoto, Japan) with the fluorophore Alexa Fluor(R) 488 (Alexa-488-silodosin) to visualize alpha(1L)-AR expression. MATERIALS AND METHODS: Radioligand binding and functional bioassay experiments were done to assess alpha(1)-AR expression in Chinese hamster ovary cells and human prostate tissues. Confocal imaging was subsequently performed. RESULTS: Although Alexa-488-silodosin had about 10 times lower affinity for all alpha(1)-AR subtypes than silodosin in binding and functional studies, it had high selectivity to alpha(1A) and alpha(1L)-ARs. Confocal imaging revealed clear localization of fluorescence on the membrane of Chinese hamster ovary cells expressing alpha(1A)-AR but not alpha(1B)-and alpha(1D)-ARs, and in the muscle layer of the human prostate. The fluorescent signal in Chinese hamster ovary cells disappeared in the presence of 3 nM prazosin but fluorescence was observed in the human prostate even in the presence of 100 nM prazosin. CONCLUSIONS: Alexa-488-silodosin is a powerful fluorescent probe with high selectivity to alpha(1A) and alpha(1L)-ARs. Thus, Alexa-488-silodosin successfully visualizes the site of alpha(1L)-ARs in the muscle layer of the human prostate without losing its distinct pharmacological profile.


Subject(s)
Fluorobenzenes , Indoles , Prostate/chemistry , Prostate/metabolism , Receptors, Adrenergic, alpha-1/analysis , Receptors, Adrenergic, alpha-1/metabolism , Animals , CHO Cells , Cricetinae , Cricetulus , Fluorescence , Male , Tissue Distribution
11.
J Pharmacol Sci ; 110(3): 389-96, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19574725

ABSTRACT

[(3)H]-CGP12177 biphasically bound to beta-adrenoceptors with high and low affinities in the segments and crude membranes of rabbit left ventricle. The low-affinity sites for [(3)H]-CGP12177 in the segments was double in density, compared to the density of high-affinity sites. Total abundance of the beta-adrenoceptors decreased to approximately 10% upon tissue homogenization, and the proportion of low-affinity sites was the same as that of the high-affinity sites in the membranes. The majority of the high-affinity binding sites of [(3)H]-CGP12177 in the segments and the membranes were beta(1H)-adrenoceptor, being highly sensitive to propranolol and beta(1)-antagonists (atenolol and ICI-89,406), whereas the low-affinity binding sites showed a beta(1L)-profile (less sensitive to propranolol and beta(1)-, beta(2)-, and beta(3)-antagonists). Furthermore, a part of the beta(1L)-adrenoceptors was insensitive to atenolol, ICI-89,406, and/or isoproterenol. The present binding study clearly shows that beta(1L)-adrenoceptors occur as a distinct phenotype from beta(1H)-adrenoceptors in rabbit ventricle. However, quantitative imbalance between beta(1H)- and beta(1L)-adrenoceptors and divergent ligand-beta(1L)-adrenoceptor interactions suggest a possibility that the beta(1L)-adrenoceptor may not reflect a simple conformational change or allosteric state in the beta(1)-adrenoceptor molecule.


Subject(s)
Heart Ventricles/chemistry , Radioligand Assay/methods , Receptors, Adrenergic, beta-1/analysis , Animals , Binding Sites , Ligands , Propanolamines/metabolism , Rabbits
12.
Eur J Neurosci ; 29(7): 1348-56, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19344328

ABSTRACT

Posttranslational modification by small ubiquitin-like modifier (SUMO) proteins is emerging as an important regulatory mechanism for neuronal function and dysfunction. Although multiple potential presynaptic SUMOylation substrate proteins have been proposed from sequence analysis the functional consequences of presynaptic SUMOylation have not been determined. Here we show that SUMOylation of presynaptic proteins modulates neurotransmitter release. Increasing protein SUMOylation by entrapping recombinant SUMO-1 in synaptosomes decreased glutamate release evoked by KCl whereas decreasing SUMOylation with the SUMO-specific protease SENP-1 enhanced KCl-evoked release. In contrast, SUMO increased and SENP-1 decreased synaptosomal glutamate release evoked by kainate stimulation. Consistent with these results, SENP-1 increased Ca(2+) influx into synaptosomes evoked by KCl whereas it decreased kainate-induced Ca(2+) influx. These results demonstrate that, in addition to postsynaptic effects, protein SUMOylation acts to modulate neurotransmitter release and thereby regulate synaptic function.


Subject(s)
Calcium/metabolism , Glutamic Acid/metabolism , Presynaptic Terminals/physiology , Small Ubiquitin-Related Modifier Proteins/metabolism , Synaptic Transmission/physiology , Animals , Central Nervous System Stimulants/administration & dosage , Endopeptidases/metabolism , Excitatory Amino Acid Agonists/administration & dosage , Kainic Acid/administration & dosage , Male , N-Methylaspartate/administration & dosage , Potassium Chloride/administration & dosage , Presynaptic Terminals/drug effects , Rats , Rats, Wistar , Recombinant Proteins/metabolism , SUMO-1 Protein/metabolism , Synaptic Transmission/drug effects , Synaptosomes/drug effects , Synaptosomes/physiology , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/administration & dosage
13.
Neurosci Lett ; 452(2): 106-10, 2009 Mar 13.
Article in English | MEDLINE | ID: mdl-19154774

ABSTRACT

GPCR interacting scaffold protein (GISP) is a multi-domain brain-specific scaffold protein that can regulate GABA(B) receptor complexes by both enhancing their surface expression and by inhibiting their lysosomal degradation. GISP retards degradation of GABA(B) receptors through its interaction with tumour susceptibility gene 101 (TSG101), a member of the endosomal sorting complex required for transport (ESCRT) lysosomal sorting machinery. We show that in addition to GABA(B), GISP exerts a more general role to increase the steady-state levels of several neurotransmitter receptors. Further, GISP delays TSG101-dependent agonist-induced EGFR down-regulation in human embryonic kidney (HEK) 293 cells whereas a mutant GISP lacking the TSG101 binding domain has no effect. These data suggest that GISP acts as a negative regulator of TSG101-dependent lysosomal degradation and plays an important role in determining the availability of neurotransmitter receptors.


Subject(s)
DNA-Binding Proteins/metabolism , Endosomes/metabolism , Lysosomes/metabolism , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Receptors, GABA-B/metabolism , Receptors, Neurotransmitter/metabolism , Transcription Factors/metabolism , A Kinase Anchor Proteins , Cell Line , Cytoskeletal Proteins , Down-Regulation/physiology , Endosomal Sorting Complexes Required for Transport , Humans , Mutation/genetics , Nerve Tissue Proteins/genetics , Protein Structure, Tertiary/genetics
14.
J Biol Chem ; 283(52): 36435-40, 2008 Dec 26.
Article in English | MEDLINE | ID: mdl-18955488

ABSTRACT

Kainate receptors (KARs) are crucial for the regulation of both excitatory and inhibitory neurotransmission, but little is known regarding the mechanisms controlling KAR surface expression. We used super ecliptic pHluorin (SEP)-tagged KAR subunit GluR6a to investigate real-time changes in KAR surface expression in hippocampal neurons. Sindbis virus-expressed SEP-GluR6 subunits efficiently co-assembled with native KAR subunits to form heteromeric receptors. Diffuse surface-expressed dendritic SEP-GluR6 is rapidly internalized following either N-methyl-d-aspartate or kainate application. Sustained kainate or transient N-methyl-d-aspartate application resulted in a slow decrease of base-line surface KAR levels. Surprisingly, however, following the initial loss of surface receptors, a short kainate application caused a long lasting increase in surface-expressed KARs to levels significantly greater than those prior to the agonist challenge. These data suggest that after initial endocytosis, transient agonist activation evokes increased KAR exocytosis and reveal that KAR surface expression is bidirectionally regulated. This process may provide a mechanism for hippocampal neurons to differentially adapt their physiological responses to changes in synaptic activation and extrasynaptic glutamate concentration.


Subject(s)
Cell Membrane/metabolism , Gene Expression Regulation , Hippocampus/metabolism , Neurons/metabolism , Receptors, Kainic Acid/biosynthesis , Animals , Glutamic Acid/metabolism , Hydrogen-Ion Concentration , Kainic Acid/metabolism , Microscopy, Confocal , Models, Biological , N-Methylaspartate/pharmacology , Receptors, Kainic Acid/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Sindbis Virus/metabolism , GluK2 Kainate Receptor
15.
J Neurochem ; 107(1): 86-95, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18643869

ABSTRACT

The neuron-specific G protein-coupled receptor interacting scaffold protein (GISP) is a multidomain, brain-specific protein derived from the A-kinase anchoring protein-9 gene. We originally isolated GISP as an interacting partner for the GABA(B) receptor subunit GABA(B1). Here, we show that the protein tumour susceptibility gene 101 (TSG101), an integral component of the endosomal sorting machinery that targets membrane proteins for lysosomal degradation, also interacts with GISP. TSG101 co-immunoprecipitates with GISP from adult rat brain, and using GST pull-downs, we identified that the eighth coiled-coiled region of GISP is critical for TSG101 association. Intriguingly, although there is no direct interaction between GISP and the GABA(B2) subunit, their co-expression in HEK293 cells increases levels of GABA(B2). GISP also inhibits TSG101-dependent GABA(B2) down-regulation in human embryonic kidney 293 cells whereas over-expression of a mutant GISP lacking the TSG101 binding domain has no effect on GABA(B2) degradation. These data suggest that GISP can function as a negative regulator of TSG101-dependent lysosomal degradation of transmembrane proteins in neurons to promote receptor stability.


Subject(s)
Brain/metabolism , DNA-Binding Proteins/metabolism , Lysosomes/metabolism , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Receptors, GABA-A/metabolism , Receptors, GABA-B/metabolism , Transcription Factors/metabolism , A Kinase Anchor Proteins , Animals , Cell Line , Cells, Cultured , Cytoskeletal Proteins , Down-Regulation/physiology , Endosomal Sorting Complexes Required for Transport , Endosomes/metabolism , Humans , Membrane Proteins/metabolism , Mutation/genetics , Nerve Tissue Proteins/genetics , Protein Binding/physiology , Protein Transport/physiology , Rats , gamma-Aminobutyric Acid/metabolism
16.
Neurosci Lett ; 436(2): 239-44, 2008 May 09.
Article in English | MEDLINE | ID: mdl-18400391

ABSTRACT

SUMOylation is emerging as an important mechanism for modulating protein function in many cell types. A large variety of proteins have been proposed as SUMO targets based on the presence of a consensus SUMOylation core motif (Psi-K-x-D/E). In neurons these include multiple synaptic proteins but it has not been established whether proteins carrying this motif are SUMOylated either in vitro or in vivo. Here we use a bacterial SUMOylation assay to systematically test for SUMO-1 modification of a selection of neuronal proteins containing one or more amino acid sequences predicted as high-probability SUMOylation sites in computer-based searches. Of the 39 proteins analysed only 14 sites were posttranslationally modified by SUMO-1, including the group III metabotropic glutamate receptors and the kainate receptor subunit GluR7. These results identify new candidate proteins that may be involved in the SUMO regulation of synaptic activity and also demonstrate that the presence of the Psi-K-x-D/E motif is not sufficient to indicate that a protein can be SUMOylated in this bacterial system.


Subject(s)
Consensus Sequence/physiology , Nerve Tissue Proteins/metabolism , Protein Processing, Post-Translational , SUMO-1 Protein/physiology , Amino Acid Motifs , Amino Acid Sequence , Animals , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , SUMO-1 Protein/chemistry , Transcription, Genetic/physiology
17.
Nat Rev Neurosci ; 8(12): 948-59, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17987030

ABSTRACT

Post-translational protein modifications are integral components of signalling cascades that enable cells to efficiently, rapidly and reversibly respond to extracellular stimuli. These modifications have crucial roles in the CNS, where the communication between neurons is particularly complex. SUMOylation is a post-translational modification in which a member of the small ubiquitin-like modifier (SUMO) family of proteins is conjugated to lysine residues in target proteins. It is well established that SUMOylation controls many aspects of nuclear function, but it is now clear that it is also a key determinant in many extranuclear neuronal processes, and it has also been implicated in a wide range of neuropathological conditions.


Subject(s)
Cell Nucleus/pathology , Cell Nucleus/physiology , Neurons/metabolism , Neurons/pathology , Protein Processing, Post-Translational/physiology , Small Ubiquitin-Related Modifier Proteins/physiology , Amino Acid Sequence , Animals , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Humans , Molecular Sequence Data , Neurons/chemistry , Neurons/physiology , Small Ubiquitin-Related Modifier Proteins/metabolism
18.
Mol Cell Neurosci ; 35(2): 339-55, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17467288

ABSTRACT

Central to synaptic function are protein scaffolds associated with neurotransmitter receptors. Alpha7 neuronal nicotinic acetylcholine receptors (nAChRs) modulate network activity, neuronal survival and cognitive processes in the CNS, but protein scaffolds that interact with these receptors are unknown. Here we show that the PDZ-domain containing protein PICK1 binds to alpha7 nAChRs and plays a role in their clustering. PICK1 interacted with the alpha7 cytoplasmic loop in yeast in a PDZ-dependent way, and the interaction was confirmed in recombinant pull-down experiments and by co-precipitation of native proteins. Some alpha7 and PICK1 clusters were adjacent at the surface of SH-SY5Y cells and GABAergic interneurons in hippocampal cultures. Expression of PICK1 caused decreased alpha7 clustering on the surface of the interneurons in a PDZ-dependent way. These data show that PICK1 negatively regulates surface clustering of alpha7 nAChRs on hippocampal interneurons, which may be important in inhibitory functions of alpha7 in the hippocampus.


Subject(s)
Carrier Proteins/metabolism , Nuclear Proteins/metabolism , Receptors, Nicotinic/metabolism , Animals , Cells, Cultured , Chemical Precipitation , Chlorocebus aethiops , Cytoskeletal Proteins , Embryo, Mammalian , Female , Hippocampus/cytology , Humans , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Pregnancy , Rats , Rats, Wistar , Transfection/methods , Two-Hybrid System Techniques , alpha7 Nicotinic Acetylcholine Receptor
19.
Nature ; 447(7142): 321-5, 2007 May 17.
Article in English | MEDLINE | ID: mdl-17486098

ABSTRACT

The small ubiquitin-like modifier protein (SUMO) regulates transcriptional activity and the translocation of proteins across the nuclear membrane. The identification of SUMO substrates outside the nucleus is progressing but little is yet known about the wider cellular role of protein SUMOylation. Here we report that in rat hippocampal neurons multiple SUMOylation targets are present at synapses and we show that the kainate receptor subunit GluR6 is a SUMO substrate. SUMOylation of GluR6 regulates endocytosis of the kainate receptor and modifies synaptic transmission. GluR6 exhibits low levels of SUMOylation under resting conditions and is rapidly SUMOylated in response to a kainate but not an N-methyl-D-aspartate (NMDA) treatment. Reducing GluR6 SUMOylation using the SUMO-specific isopeptidase SENP-1 prevents kainate-evoked endocytosis of the kainate receptor. Furthermore, a mutated non-SUMOylatable form of GluR6 is not endocytosed in response to kainate in COS-7 cells. Consistent with this, electrophysiological recordings in hippocampal slices demonstrate that kainate-receptor-mediated excitatory postsynaptic currents are decreased by SUMOylation and enhanced by deSUMOylation. These data reveal a previously unsuspected role for SUMO in the regulation of synaptic function.


Subject(s)
Receptors, Kainic Acid/metabolism , SUMO-1 Protein/metabolism , Synaptic Transmission , Animals , Brain/cytology , Cells, Cultured , Endocytosis/drug effects , Kainic Acid/pharmacology , Neurons/drug effects , Neurons/metabolism , Rats , Receptors, Kainic Acid/agonists , Substrate Specificity , Synapses/drug effects , Synapses/metabolism , Synaptic Transmission/drug effects , GluK2 Kainate Receptor
20.
J Neurochem ; 100(4): 1003-17, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17241134

ABSTRACT

Synaptic transmission depends on the regulated surface expression of neurotransmitter receptors, but many of the cellular processes required to achieve this remain poorly understood. To better define specific mechanisms for the GABA(B) receptor (GABA(B)R) trafficking, we screened for proteins that bind to the carboxy-terminus of the GABA(B1) subunit. We report the identification and characterization of a novel 130-kDa protein, GPCR interacting scaffolding protein (GISP), that interacts directly with the GABA(B1) subunit via a coiled-coil domain. GISP co-fractionates with GABA(B)R and with the postsynaptic density and co-immunoprecipitates with GABA(B1) and GABA(B2) from rat brain. In cultured hippocampal neurons, GISP displays a punctate dendritic distribution and has an overlapping localization with GABA(B)Rs. When co-expressed with GABA(B)Rs in human embryonic kidney cells, GISP promotes GABA(B)R surface expression and enhances both baclofen-evoked extracellular signal-regulated kinase (ERK) phosphorylation and G-protein inwardly rectifying potassium channel (GIRK) currents. These results suggest that GISP is involved in the forward trafficking and stabilization of functional GABA(B)Rs.


Subject(s)
Cytoskeletal Proteins/physiology , Gene Expression Regulation/drug effects , Receptors, GABA-B/physiology , A Kinase Anchor Proteins , Animals , Baclofen/pharmacology , Biotinylation/methods , Cells, Cultured , Embryo, Mammalian , GABA Agonists/pharmacology , Gene Expression Regulation/physiology , Hippocampus/cytology , Humans , Immunoprecipitation/methods , Membrane Potentials/drug effects , Membrane Potentials/physiology , Molecular Weight , Mutagenesis/physiology , Nerve Tissue Proteins/physiology , Neurons/drug effects , Neurons/metabolism , Protein Structure, Tertiary/physiology , Protein Transport/drug effects , Protein Transport/physiology , Rats , Receptors, GABA-B/chemistry , Subcellular Fractions/drug effects , Subcellular Fractions/physiology , Transfection , Two-Hybrid System Techniques
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