A carboxyl-terminal determinant of the neuronal glutamate transporter, EAAC1, is required for platelet-derived growth factor-dependent trafficking.
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.
Sheldon
Amanda L
AL
Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, Pennsylvania 19104-4318, USA.
González
Marco I
MI
Robinson
Michael B
MB
D000409:Alanine / Q000737:chemistry; D020816:Amino Acid Motifs; D000818:Animals; D001692:Biological Transport; D019921:Biotinylation; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D027342:Excitatory Amino Acid Transporter 2 / Q000378:metabolism; D050580:Excitatory Amino Acid Transporter 3 / Q000502:physiology*; D018698:Glutamic Acid / Q000378:metabolism*; D047468:Immunoprecipitation; D016296:Mutagenesis; D009154:Mutation; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012964:Sodium / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry
D018076:DNA, Complementary; D027342:Excitatory Amino Acid Transporter 2; D050580:Excitatory Amino Acid Transporter 3; D010982:Platelet-Derived Growth Factor; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D018698:Glutamic Acid; D014443:Tyrosine; D012964:Sodium; D000409:Alanine