Rapid trafficking of the neuronal glutamate transporter, EAAC1: evidence for distinct trafficking pathways differentially regulated by protein kinase C and platelet-derived growth factor.
The neuronal glutamate transporter, EAAC1, appears to both limit spillover between excitatory synapses and provide precursor for the synthesis of the inhibitory neurotransmitter, gamma-aminobutyric acid. There is evidence for a large intracellular pool of EAAC1 from which transporter is redistributed to the cell surface following activation of protein kinase C (PKC) or platelet-derived growth factor (PDGF) receptor by seemingly independent pathways. A variety of biotinylation strategies were employed to measure trafficking of EAAC1 to and from the plasma membrane and to examine the effects of phorbol ester and PDGF on these events. Biotinylation of cell surface protein under trafficking-permissive conditions (37 degrees C) resulted in a 2-fold increase in the amount of biotinylated EAAC1 within 15 min in C6 glioma and in primary neuronal cultures, suggesting that EAAC1 has a half-life of approximately 5-7 min for residence at the plasma membrane. Both phorbol ester and PDGF increased the amount of transporter labeled under these conditions. Using a reversible biotinylation strategy, a similarly rapid internalization of EAAC1 was observed in C6 glioma. Phorbol ester, but not PDGF, blocked this measure of internalization. Incubation at 18 degrees C, which blocks some forms of intracellular membrane trafficking, inhibited PKC- and PDGF-dependent redistribution of EAAC1 but had no effect on basal trafficking of EAAC1. These studies suggest that both PKC and PDGF accelerate delivery of EAAC1 to the cell surface and that PKC has an additional effect on endocytosis. The data also suggest that basal and regulated pools of EAAC1 exist in distinct compartments.
Fournier
Keith M
KM
Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania, 3615 Civic Center Boulevard, Philadelphia, PA 19104-4318, USA.
González
Marco I
MI
Robinson
Michael B
MB
D027322:Amino Acid Transport System X-AG / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D019921:Biotinylation; D015153:Blotting, Western; D001921:Brain / Q000196:embryology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004705:Endocytosis; D050580:Excitatory Amino Acid Transporter 3; D015971:Gene Expression Regulation, Enzymologic; D050491:Glutamate Plasma Membrane Transport Proteins; D009474:Neurons / Q000378:metabolism; D010982:Platelet-Derived Growth Factor; D011493:Protein Kinase C / Q000378:metabolism*; D021381:Protein Transport; D051381:Rats; D012964:Sodium / Q000378:metabolism; D027981:Symporters / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors; D014162:Transfection
D027322:Amino Acid Transport System X-AG; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D010982:Platelet-Derived Growth Factor; C493398:Slc1a1 protein, rat; D027981:Symporters; D012964:Sodium; D011493:Protein Kinase C