The four major N- and C-terminal splice variants of the excitatory amino acid transporter GLT-1 form cell surface homomeric and heteromeric assemblies.
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.
Peacey
Eleanor
E
Wolfson Centre for Age-Related Diseases, King's College London, London, United Kingdom.
Miller
Christopher C J
CC
Dunlop
John
J
Rattray
Marcus
M
D000818:Animals; D002478:Cells, Cultured; D027342:Excitatory Amino Acid Transporter 2 / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D006801:Humans; D051379:Mice; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D027342:Excitatory Amino Acid Transporter 2; D020033:Protein Isoforms; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate