title: Internalization and degradation of the glutamate transporter GLT-1 in response to phorbol ester.
abstract: Activation of protein kinase C (PKC) decreases the activity and cell surface expression of the predominant forebrain glutamate transporter, GLT-1. In the present study, C6 glioma were used as a model system to define the mechanisms that contribute to this decrease in cell surface expression and to determine the fate of internalized transporter. As was previously observed, phorbol 12-myristate 13-acetate (PMA) caused a decrease in biotinylated GLT-1. This effect was blocked by sucrose or by co-expression with a dominant-negative variant of dynamin 1, and it was attenuated by co-expression with a dominant-negative variant of the clathrin heavy chain. Depletion of cholesterol with methyl-beta-cyclodextrin, co-expression with a dominant-negative caveolin-1 mutant (Cav1/S80E), co-expression with dominant-negative variants of Eps15 (epidermal-growth-factor receptor pathway substrate clone 15), or co-expression with dominant-negative Arf6 (T27N) had no effect on the PMA-induced loss of biotinylated GLT-1. Long-term treatment with PMA caused a time-dependent loss of biotinylated GLT-1 and decreased the levels of GLT-1 protein. Inhibitors of lysosomal degradation (chloroquine or ammonium chloride) or co-expression with a dominant-negative variant of a small GTPase implicated in trafficking to lysosomes (Rab7) prevented the PMA-induced decrease in protein and caused an intracellular accumulation of GLT-1. These results suggest that the PKC-induced redistribution of GLT-1 is dependent upon clathrin-mediated endocytosis. These studies identify a novel mechanism by which the levels of GLT-1 could be rapidly down-regulated via lysosomal degradation. The possibility that this mechanism may contribute to the loss of GLT-1 observed after acute insults to the CNS is discussed.
authors.affiliation: Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania, 502N Abramson Pediatric Research Building, 3615 Civic Center Blvd., Philadelphia, PA 19104-4318, United States.
authors.forename: Bala T S
authors.initials: BT
authors.lastname: Susarla
authors.forename: Michael B
authors.initials: MB
authors.lastname: Robinson
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D034281:Dynamins / Q000378:metabolism; D027342:Excitatory Amino Acid Transporter 2 / Q000378:metabolism*; D006801:Humans; D008247:Lysosomes / Q000187:drug effects / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D013395:Sucrose / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection; D020691:rab GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D000090802:rab7 GTP-Binding Proteins
chemical_list: D000089982:ADP-Ribosylation Factor 6; D002966:Clathrin; D018076:DNA, Complementary; D027342:Excitatory Amino Acid Transporter 2; D008565:Membrane Proteins; D000090802:rab7 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human; D013395:Sucrose; D001710:Biotin; D002784:Cholesterol; D011493:Protein Kinase C; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; D020691:rab GTP-Binding Proteins; D034281:Dynamins; D013755:Tetradecanoylphorbol Acetate