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Modulation of the neuronal glutamate transporter EAAT4 by two interacting proteins

Abstract

Glutamate is the main excitatory neurotransmitter in the mammalian central nervous system and is removed from the synaptic cleft by sodium-dependent glutamate transporters. To date, five distinct glutamate transporters have been cloned from animal and human tissue: GLAST (EAAT1), GLT-1 (EAAT2), EAAC1 (EAAT3), EAAT4, and EAAT5 (refs 1,2,3,4,5). GLAST and GLT-1 are localized primarily in astrocytes6,7, whereas EAAC1 (refs 8, 9), EAAT4 (refs 9,10,11) and EAAT5 (ref. 5) are neuronal. Studies of EAAT4 and EAAC1 indicate an extrasynaptic localization on perisynaptic membranes that are near release sites8,9,10. This localization facilitates rapid glutamate binding, and may have a role in shaping the amplitude of postsynaptic responses in densely packed cerebellar terminals12,13,14,15. We have used a yeast two-hybrid screen to identify interacting proteins that may be involved in regulating EAAT4—the glutamate transporter expressed predominately in the cerebellum—or in targeting and/or anchoring or clustering the transporter to the target site. Here we report the identification and characterization of two proteins, GTRAP41 and GTRAP48 (for glutamate transporter EAAT4 associated protein) that specifically interact with the intracellular carboxy-terminal domain of EAAT4 and modulate its glutamate transport activity.

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Figure 1: Structure and distribution of GTRAP41 and GTRAP48. a, b, cDNA clones GTRAP41/pGAD10 and GTRAP48/pGAD10 isolated from the yeast two-hybrid screen are shown aligned below representations of full-length GTRAP41 and GTRAP48, respectively.
Figure 2: Interaction of GTRAP41 and GTRAP48 with EAAT4.
Figure 3: Guanine nucleotide exchange activity of GTRAP48.
Figure 4: Effect of GTRAP41 and GTRAP48 on Na+-dependent [3H]l-glutamate uptake.
Figure 5: Modulation of EAAT4 activity.

Accession codes

Accessions

GenBank/EMBL/DDBJ

Data deposits

The GenBank accession numbers are AF225960 (GTRAP41) and AF225961 (GTRAP48).

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Acknowledgements

We thank N. J. Maragakis, M. Watanabe, A. Sawa, R. Ganel, J. Llado and R. Law for discussions, advice and help. We thank R. Huganir for the pRK5 vector and D. Howard for technical assistance. This work was supported by the NIH.

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Correspondence to Jeffrey D. Rothstein.

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Jackson, M., Song, W., Liu, MY. et al. Modulation of the neuronal glutamate transporter EAAT4 by two interacting proteins. Nature 410, 89–93 (2001). https://doi.org/10.1038/35065091

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