Ubiquitination-mediated internalization and degradation of the astroglial glutamate transporter, GLT-1.
Sodium-dependent glutamate uptake is essential for limiting excitotoxicity, and dysregulation of this process has been implicated in a wide array of neurological disorders. The majority of forebrain glutamate uptake is mediated by the astroglial glutamate transporter, GLT-1. We and others have shown that this transporter undergoes endocytosis and degradation in response to activation of protein kinase C (PKC), however, the mechanisms involved remain unclear. In the current study, transfected C6 glioma cells or primary cortical cultures were used to show that PKC activation results in incorporation of ubiquitin into GLT-1 immunoprecipitates. Mutation of all 11 lysine residues in the amino and carboxyl-terminal domains to arginine (11R) abolished this signal. Selective mutation of the seven lysine residues in the carboxyl terminus (C7K-R) did not eliminate ubiquitination, but it completely blocked PKC-dependent internalization and degradation. Two families of variants of GLT-1 were prepared with various lysine residues mutated to arginine. Analyses of these constructs indicated that redundant lysine residues in the carboxyl terminus were sufficient for the appearance of ubiquitinated product and degradation of GLT-1. Together these data define a novel mechanism by which the predominant forebrain glutamate transporter can be rapidly targeted for degradation.
Sheldon
Amanda L
AL
Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA 19104, USA.
González
Marco I
MI
Krizman-Genda
Elizabeth N
EN
Susarla
Bala T S
BT
Robinson
Michael B
MB
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D045744:Cell Line, Tumor; D002540:Cerebral Cortex / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics; D004705:Endocytosis / Q000502:physiology; D027342:Excitatory Amino Acid Transporter 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology*
D027342:Excitatory Amino Acid Transporter 2; D025801:Ubiquitin; D018698:Glutamic Acid; D011493:Protein Kinase C; D008239:Lysine