Differential regulation of GLAST immunoreactivity and activity by protein kinase C: evidence for modification of amino and carboxyl termini.
Many neurotransmitter transporters, including the GLT-1 and EAAC1 subtypes of the glutamate transporter, are regulated by protein kinase C (PKC) and these effects are associated with changes in cell surface expression. In the present study, the effects of PKC activation on the glutamate aspartate transporter (GLAST) subtype of glutamate transporter were examined in primary astrocyte cultures. Acute (30 min) exposure to the phorbol 12-myristate 13-acetate (PMA) increased (approximately 20%) transport activity but had the opposite effect on both total and cell surface immunoreactivity. Chronic treatment (6 or 24 h) with PMA had no effect on transport activity but caused an even larger decrease in total and cell surface immunoreactivity. This loss of immunoreactivity was observed using antibodies directed against three different cytoplasmic epitopes, and was blocked by the PKC antagonist, bisindolylmaleimide II. We provide biochemical and pharmacological evidence that the activity observed after treatment with PMA is mediated by GLAST. Two different flag-tagged variants of the human homolog of GLAST were introduced into astrocytes using lentiviral vectors. Although treatment with PMA caused a loss of transporter immunoreactivity, flag immunoreactivity did not change in amount or size. Together, these studies suggest that activation of PKC acutely up-regulates GLAST activity, but also results in modification of several different intracellular epitopes so that they are no longer recognized by anti-GLAST antibodies. We found that exposure of primary cultures of neurons/astrocytes to transient hypoxia/glucose deprivation also caused a loss of GLAST immunoreactivity that was attenuated by the PKC antagonist, bisindolylmaleimide II, suggesting that some acute insults previously thought to cause a loss of GLAST protein may mimic the phenomenon observed in the present study.
Susarla
Bala T S
BT
Department of Pediatrics, University of Pennsylvania and Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Seal
Rebecca P
RP
Zelenaia
Olga
O
Watson
Deborah J
DJ
Wolfe
John H
JH
Amara
Susan G
SG
Robinson
Michael B
MB
D027322:Amino Acid Transport System X-AG / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology / Q000378:metabolism*; D015153:Blotting, Western / Q000379:methods; D015687:Cell Hypoxia; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D018920:Coculture Techniques / Q000379:methods; D004347:Drug Interactions; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000939:Epitopes / Q000378:metabolism; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2 / Q000378:metabolism; D050580:Excitatory Amino Acid Transporter 3; D005947:Glucose / Q000172:deficiency; D050491:Glutamate Plasma Membrane Transport Proteins; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D010703:Phorbol Esters / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D012964:Sodium / Q000378:metabolism; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D027981:Symporters / Q000378:metabolism; D013997:Time Factors; D014162:Transfection / Q000379:methods
D027322:Amino Acid Transport System X-AG; D004791:Enzyme Inhibitors; D000939:Epitopes; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D007211:Indoles; D008301:Maleimides; D010703:Phorbol Esters; C493400:SLC1A1 protein, human; C493394:SLC1A3 protein, human; C493398:Slc1a1 protein, rat; C493392:Slc1a3 protein, rat; D027981:Symporters; C008874:phorbol-12,13-diacetate; D018698:Glutamic Acid; D012964:Sodium; D011493:Protein Kinase C; D005947:Glucose; C070515:bisindolylmaleimide I