Platelet-derived Growth Factor Rapidly Increases Activity and Cell Surface Expression of the EAAC1 Subtype of Glutamate Transporter through Activation of Phosphatidylinositol 3-Kinase

Karen D Sims,Dean J Straff,Michael B Robinson

doi:10.1074/jbc.275.7.5228

Abstract

Na(+)-dependent glutamate transporters are the primary mechanism for removal of excitatory amino acids (EAAs) from the extracellular space of the central nervous system and influence both physiologic and pathologic effects of these compounds. Recent evidence suggests that the activity and cell surface expression of a neuronal subtype of glutamate transporter, EAAC1, are rapidly increased by direct activation of protein kinase C and are decreased by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K). We hypothesized that this regulation could be analogous to insulin-induced stimulation of the GLUT4 subtype of glucose transporter, which is dependent upon activation of PI3-K. Using C6 glioma, a cell line that endogenously and selectively expresses EAAC1, we report that platelet-derived growth factor (PDGF) increased Na(+)-dependent L-[(3)H]-glutamate transport activity within 30 min. This effect of PDGF was not due to a change in total cellular EAAC1 immunoreactivity but was instead correlated with an increase cell surface expression of EAAC1, as measured using a membrane impermeant biotinylation reagent combined with Western blotting. A decrease in nonbiotinylated intracellular EAAC1 was also observed. These studies suggest that PDGF causes a redistribution of EAAC1 from an intracellular compartment to the cell surface. These effects of PDGF were accompanied by a 35-fold increase in PI3-K activity and were blocked by the PI3-K inhibitors, wortmannin and LY 294002, but not by an inhibitor of protein kinase C. Other growth factors, including insulin, nerve growth factor, and epidermal growth factor had no effect on glutamate transport nor did they increase PI3-K activity. These studies suggest that, as is observed for insulin-mediated translocation of GLUT4, EAAC1 cell surface expression can be rapidly increased by PDGF through activation of PI3-K. It is possible that this PDGF-mediated increase in EAAC1 activity may contribute to the previously demonstrated neuroprotective effects of PDGF.

Highlights

The rapid clearance of glutamate from the extracellular space of the central nervous system by Naϩ-dependent high affinity glutamate transporters is critical to the maintenance of effective synaptic transmission and the prevention of excitotoxic injury
Glioma can be rapidly regulated by exogenous pharmacological agents, but it is not known if endogenous central nervous system substances can induce receptor-mediated changes in activity or trafficking
We hypothesized that growth factors may regulate the activity and cell surface expression of EAAC1 as is observed for insulin-dependent regulation of the GLUT4 subtype of glucose transporter

Summary

Introduction

The rapid clearance of glutamate from the extracellular space of the central nervous system by Naϩ-dependent high affinity glutamate transporters is critical to the maintenance of effective synaptic transmission and the prevention of excitotoxic injury. Recent studies demonstrate that the activity of several neurotransmitter transporters can be rapidly regulated by direct activation of intracellular signaling molecules (PKC or cAMPdependent protein kinase), including norepinephrine [20], serotonin [21], dopamine [22,23,24], GABA [25, 26], and glutamate transporters [27]. Recent studies have demonstrated that activation of G protein-coupled receptors causes a decrease in cell surface expression of the GAT1 subtype of GABA transporter in neurons [32]. We recently demonstrated that activation of PKC with phorbol ester increases the activity and cell surface expression of the EAAC1 subtype of glutamate transporter [27]. We found that wortmannin, an inhibitor of PI3-K, decreased EAAC1 activity and cell surface expression These regulated changes in activity and cell surface expression qualitatively resemble translocation events observed for the insulin-sensitive glucose transporter GLUT4 The regulation of GLUT4 has been well studied and appears to be primarily mediated by activation of the insulin receptor tyrosine kinase cascade and stimulation of PI3-K, some studies suggest an additional role for phorbol 12-myristate 13-acetate (PMA)-sensitive PKCs

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