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.Excitatory neurotransmission in the mammalian central nervous system is mediated predominantly by the acidic amino acid glutamate. In addition to mediating swift synaptic depolarization, glutamatergic neurotransmission is important for physiological processes such as synapse development and synaptic plasticity (1-3). However, high levels of extracellular glutamate result in excessive activation of glutamate receptors, a factor that presumably contributes to the cytotoxicity associated with stroke, hypoglycemia, and brain injury (4 -6). Synaptic glutamate concentrations are tightly regulated by a family of high affinity, Na ϩ -dependent glutamate transporters, ensuring crisp synaptic neurotransmission. This family consists of five members: GLAST 4 (EAAT1), GLT-1 (EAAT2), EAAC1 (EAAT3), EAAT4, and EAAT5 (for review, see Refs. 7 and 8). In contrast to other neurotransmitter transporters, EAAC1 is expressed predominantly on postsynaptic neurons, where it is localized to perisynaptic membrane (9, 10). Here, uptake via EAAC1 is believed to prevent glutamate spillover and activation of extrasynaptic receptors (11). Antisense knock-down of the neuronal glutamate transporter revealed that transport by EAAC1 accounts for ϳ40% of glutamate uptake in the hippocampus (12), an area that is both extremely plastic and susce...