Regulation of excitatory amino acid transporters by the associated protein GTRAP3-18

High affinity excitatory amino acid transporters (EAAT) terminate glutamatergic synaptic transmission at synaptic boutons within the nervous system. The EAAT gene family consists of five structurally conserved isoforms with specific expression within astrocytic, neuronal and additional cell types. We performed a yeast-two-hybrid screen to identify intracellular binding partners for the carboxyl terminus of the most abundant neuronal transporter, EAAC1/EAAT3. We characterized a 22kD protein isolated from that screen that we named GTRAP3-18 (G&barbelow;lutamate Transporter A&barbelow;ssociated P&barbelow;rotein of EAAT3&barbelow;). GTRAP3-18 has the physiologic effect of decreasing EAACI activity in-vivo and in-vitro.; GTRAP3-18 is a resident endoplasmic reticulum (ER) protein. EAAC1 association with GTRAP3-18 decreases the rate of trafficking from the ER, resulting in accumulation within ER structures. GTRAP3-18 alters the activity of EAAC1 through a decrease in the Vmax of transport and an increase in the measured Km for glutamate. Antisense reduction in GTRAP or removal of the C-terminal interaction sight causes a reversal of these alterations to EAAC1. The locations of extra-cellular N-linked glycosylation sites are conserved and required for ER protein assembly and export. We found that N178 is necessary for EAAC1 to be properly glycosylated and expressed on the plasma membrane, while N128 and N194 contribute to EAAC1 protein stability and efficient surface expression.; The perturbations to transporter physiology and protein maturation following GTRAP3-18 co-expression are similarly found with all the other EAAT isoforms and the related ASCT family. GTRAP3-18 is further able to reduce the maturation of the Na+/Cl- family of neurotransmitter transporters and the G-protein coupled receptor family. GTRAP3-18 does not alter trafficking of membrane associated proteins, single transmembrane spanning proteins, or proteins targeted to areas in the cell other than the plasma membrane. The GTRAP gene is highly conserved throughout species and has been shown to be induced in response to ER stress. GTRAP3-18 mediates the dynamics of subunit assembly of nascent multi-subunit oligomeric proteins before ER exit in COPII vesicles and may act as an inducible component of the UPR (unfolded protein response) system in cells.