Inhibition of the high-affinity brain glutamate transporter GLAST-1 via direct phosphorylation.
Neurotransmission at excitatory glutamatergic synapses is terminated by the reuptake of the neurotransmitter by high-affinity transporters, which keep the extracellular glutamate concentration below excitotoxic levels. The amino acid sequence of the recently isolated and cloned brain-specific glutamate/aspartate transporter (GLAST-1) of the rat reveals three consensus sequences of putative phosphorylation sites for protein kinase C (PKC). The PKC activator phorbol 12-myristate 13-acetate (PMA) decreased glutamate transport activity in Xenopus oocytes and human embryonic kidney cells (HEK293) expressing the cloned GLAST-1 cDNA, within 20 min, to 25% of the initial transport activity. This downregulation was blocked by the PKC inhibitor staurosporine. GLAST-1 transport activity remains unimpaired by phorbol 12-monomyristate. Removal of all putative PKC sites of wild-type GLAST-1 by site-directed mutagenesis did not abolish inhibition of glutamate transport. [32P]Phosphate-labeled wild-type and mutant transport proteins devoid of all predicted PKC sites were detected by immunoprecipitation after stimulation with PMA. Immunoprecipitation of [35S]methionine-labeled transporter molecules indicates a similar stability of phosphorylated and nonphosphorylated GLAST-1 protein. Immunofluorescence staining did not differentiate surface staining of HEK293 cells expressing GLAST-1 with and without PMA treatment. These data suggest that the neurotransmitter transporter activity of GLAST-1 is inhibited by phosphorylation at a non-PKC consensus site.
Conradt
M
M
Institute of Biochemistry I, Medical Faculty, University of Cologne, Germany.
Stoffel
W
W
D000255:Adenosine Triphosphate / Q000378:metabolism; D027322:Amino Acid Transport System X-AG; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D005260:Female; D006023:Glycoproteins / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D009865:Oocytes / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014982:Xenopus laevis
D027322:Amino Acid Transport System X-AG; D002352:Carrier Proteins; D006023:Glycoproteins; D010710:Phosphates; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate