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-β-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 dominantnegative variant of a small GTPase implicated in trafficking to lysosomes (Rab7) prevented the PMAinduced 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 downregulated 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.

Protein Kinase C-Mediated Phosphorylation of a Single Serine Residue on the Rat Glial Glutamine Transporter SN1 Governs Its Membrane Trafficking

“…In addition, the glial GLT and GLAST are specifically targeted to small glial processes in the vicinity of synapses, and in the same processes, SN1 is also specifically enriched (Chaudhry et al, 1995;Boulland et al, 2002). Our current data show that prolonged PMA-induced stimulation results in degradation of SN1, and the time course coincides with the demonstrated internalization and subsequent degeneration of GLT (Susarla and Robinson, 2008). Thus, the glial glutamate and glutamine transporters may be targeted by similar regulatory mechanisms as a response to certain synaptic activity synergistically inhibiting glutamate recycling.…”

“…Several transporters are degraded after retrieval from the plasma membrane (Daniels and Amara, 1999;Susarla and Robinson, 2008). We therefore investigated whether the PKC-mediated internalization of SN1 is followed by degradation of the protein.…”

Glutamate Transporter Coupling to Na,K-ATPase

“…The effects of protein kinases on glutamate transporters are complex and vary considerably depending on the preparation and conditions. Protein kinase C has been shown to downregulate GLT-1 in C6 glioma cells (Susarla and Robinson, 2008), whereas other studies have shown that GLT-1 and GLAST are upregulated by growth factor-induced tyrosine kinase activation and that this effect can be blocked by the tyrosine kinase inhibitors (Zelenaia et al, 2000;Koeberle and Bähr, 2008). Ouabain has been shown to induce intracellular calcium oscillations via a direct interaction between the N-terminal tail of Na,K-ATPase and the inositol 1,4,5-trisphosphate-ligand binding domain of the IP 3 receptor (Mikoshiba, 2007), and low concentrations of ouabain can trigger slow, low-frequency calcium oscillations that activate the transcription factor nuclear factor B.…”

The Four Major N- and C-Terminal Splice Variants of the Excitatory Amino Acid Transporter GLT-1 Form Cell Surface Homomeric and Heteromeric Assemblies

“…Activation of protein kinase C (PKC) by phorbol esters has been suggested to drive an internalization of GLT-1 in a number of cell types, including stably and transiently transfected C6 glioma, primary mouse astrocytes induced to express GLT-1 by dibutyryl cAMP, and neuron-enriched rat cortical cocultures (Susarla and Robinson 2008;Zhou and Sutherland 2004;Kalandadze et al, 2002;Gonzá lez et al, 2005;Guillet et al, 2005). As a proof of concept to test the utility of these novel epitope-tagged GLT-1 isoforms to study regulated trafficking, we used HEK-293 cells stably expressing V5-MPK-DIETCI and surveyed the cell surface and intracellular expression of GLT-1 using an anti-V5 antibody.…”