Abstract
The l-glutamate transporter GLT-1 is an abundant central nervous system (CNS) membrane protein of the excitatory amino acid transporter (EAAT) family that controls extracellular l-glutamate levels and is important in limiting excitotoxic neuronal death. Using reverse transcription-polymerase chain reaction, we have determined that four mRNAs encoding GLT-1 exist in mouse brain, with the potential to encode four GLT-1 isoforms that differ in their N and C termini. We expressed all four isoforms (termed MAST-KREK, MPK-KREK, MAST-DIETCI, and MPK-DIETCI according to amino acid sequence) in a range of cell lines and primary astrocytes and show that each isoform can reach the cell surface. In transfected human embryonic kidney (HEK) 293 or COS-7 cells, all four isoforms support high-affinity sodium-dependent l-glutamate uptake with identical pharmacological and kinetic properties. Inserting a viral epitope (tagged with V5, hemagglutinin, or FLAG) into the second extracellular domain of each isoform allowed coimmunoprecipitation and time-resolved Förster resonance energy transfer (tr-FRET) studies using transfected HEK-293 cells. Here we show for the first time that each of the four isoforms is able to combine to form homomeric and heteromeric assemblies, each of which is expressed at the cell surface of primary astrocytes. After activation of protein kinase C by phorbol ester, V5-tagged GLT-1 is rapidly removed from the cell surface of HEK-293 cells and degraded. This study provides direct biochemical evidence for oligomeric assembly of GLT-1 and reports the development of novel tools to provide insight into the trafficking of GLT-1.
Footnotes
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This work was supported by the Medical Research Council [Doctoral Training Grant and Grant G0501573]; the Motor Neuron Disease Association [UK: USA collaborative award]; and the Wellcome Trust [Grant 078662].
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ABBREVIATIONS: GLT-1, glutamate transporter-1; CNS, central nervous system; ALS, amyotrophic lateral sclerosis; HA, hemagglutinin; PCR, polymerase chain reaction; FBS, fetal bovine serum; PMSF, phenylmethylsulfonyl fluoride; TBS, Tris-buffered saline; TTBS, Tris-buffered saline/Tween 20; HBS, HEPES-buffered saline solution; WAY-213394, N4-(2′-methyl-1,1′-biphenyl-4-yl)-l-asparagine; WAY-213613, N4-[4-(2-bromo-4,5-difluorophenoxy) phenyl]-l-asparagine; WAY-212922, N4-[7-(trifluoromethyl)-9H-fluoren-2-yl]-l-asparagine; WAY-144855, 3-amino-tricyclo[2.2.1.02.6]heptane-1,3-dicarboxylic acid; NGS, normal goat serum; HEK, human embryonic kidney; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; IC, intracellular; M, membrane; ANOVA, analysis of variance; tr-FRET, time-resolved Förster resonance energy transfer; PKC, protein kinase C; SOD1, superoxide dismutase-1; GFP, green fluorescent protein; EAAT, excitatory amino acid transporter.
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received October 15, 2008.
- Accepted February 6, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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