A carboxyl-terminal determinant of the neuronal glutamate transporter, EAAC1, is required for platelet-derived growth factor-dependent trafficking.

Abstract	The neuronal glutamate transporter, EAAC1 (excitatory amino acid carrier 1), undergoes rapid regulation after treatment with platelet-derived growth factor (PDGF) or phorbol ester in C6 glioma cells and neurons. A large intracellular pool of EAAC1 exists, from which transporters are redistributed to the cell surface in response to these signals. Here we show that PDGF had no effect on subcellular localization of the glial glutamate transporter, GLT-1, after transfection into C6 glioma cells. Chimeras consisting of domains from EAAC1 or GLT-1 were used to investigate structural motifs involved in PDGF-dependent redistribution of EAAC1. PDGF did not induce trafficking of an EAAC1 chimera containing the carboxyl-terminal domain of GLT-1; however, it did induce trafficking of a GLT-1 chimera containing the carboxyl-terminal domain of EAAC1. A truncated mutant of EAAC1 lacking 10 carboxyl-terminal amino acids was responsive to PDGF, whereas a mutant lacking 20 residues was not. Alanine substitution mutagenesis in this region revealed a short motif, (502)YVN(504), necessary for regulated trafficking. This motif was also involved in protein kinase C-dependent trafficking, as mutant transporters exhibited an attenuated response to phorbol ester. Interestingly, the presence of YVN in the homologous region of a nonresponsive chimera was not sufficient to confer regulated trafficking; however, the presence of a 12-amino acid motif starting at this Tyr residue was sufficient to confer responsiveness to PDGF. These studies identify a novel motif within the carboxyl terminus of EAAC1 which is required for regulated trafficking. The possibility that this motif targets EAAC1 to an intracellular, "regulated pool" is discussed.
Authors	Amanda L Sheldon, Marco I González, Michael B Robinson
Journal	The Journal of biological chemistry (J Biol Chem) Vol. 281 Issue 8 Pg. 4876-86 (Feb 24 2006) ISSN: 0021-9258 [Print] United States
PMID	16368696 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
Chemical References	DNA, Complementary Excitatory Amino Acid Transporter 2 Excitatory Amino Acid Transporter 3 Platelet-Derived Growth Factor Recombinant Fusion Proteins Recombinant Proteins Glutamic Acid Tyrosine Sodium Alanine
Topics	Alanine (chemistry) Amino Acid Motifs Animals Biological Transport Biotinylation Blotting, Western Cell Line, Tumor Cell Membrane (metabolism) Cloning, Molecular DNA, Complementary (metabolism) Excitatory Amino Acid Transporter 2 (metabolism) Excitatory Amino Acid Transporter 3 (physiology) Glutamic Acid (metabolism) Immunoprecipitation Mutagenesis Mutation Phosphorylation Platelet-Derived Growth Factor (metabolism) Protein Structure, Tertiary Rats Recombinant Fusion Proteins (chemistry) Recombinant Proteins (chemistry) Sodium (chemistry) Time Factors Transfection Tyrosine (chemistry)

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