GLAST stability and activity are enhanced by interaction with the PDZ scaffold NHERF-2.
The astrocytic glutamate transporter GLAST (also known as EAAT1) is a key regulator of extracellular glutamate levels in many regions of vertebrate brains. To identify novel interacting partners that might regulate the localization and function of GLAST in astrocytes, we screened the transporter's C-terminus (GLAST-CT) against a proteomic array of 96 different PDZ domains. The GLAST-CT robustly and specifically interacted with PDZ domains from two related scaffolding proteins, the Na(+)/H(+) exchanger regulatory factors 1 and 2 (NHERF-1 and NHERF-2). Studies on cultured rat cortical astrocytes revealed that these cells are highly enriched in NHERF-2 relative to NHERF-1. Endogenous GLAST and NHERF-2 from cultured astrocytes were found to robustly co-immunoprecipitate, and further co-immunoprecipitation studies on mutant versions of GLAST expressed in transfected cells revealed the GLAST/NHERF-2 interaction to be dependent on the last amino acid of the GLAST-CT. Knockdown of endogenous NHERF-2 in astrocytes via siRNA treatment resulted in a significant reduction in GLAST activity, which corresponded to significantly reduced total expression of GLAST protein and reduced half-life of GLAST, as assessed in pulse-chase metabolic labeling studies. These findings reveal that NHERF-2 can interact with GLAST in astrocytes to enhance GLAST stability and activity.
Ritter
Stefanie L
SL
Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322, USA.
Asay
Matthew J
MJ
Paquet
Maryse
M
Paavola
Kevin J
KJ
Reiff
Rachel E
RE
Yun
C Chris
CC
Hall
Randy A
RA
D000818:Animals; D001224:Aspartic Acid / Q000493:pharmacokinetics; D001253:Astrocytes; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D027341:Excitatory Amino Acid Transporter 1 / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D020125:Mutation, Missense / Q000235:genetics; D019579:Neocortex / Q000166:cytology; D054731:PDZ Domains / Q000187:drug effects / Q000502:physiology*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods; D014316:Tritium / Q000493:pharmacokinetics
D027341:Excitatory Amino Acid Transporter 1; D010750:Phosphoproteins; D034741:RNA, Small Interfering; C493394:SLC1A3 protein, human; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D014316:Tritium; D001224:Aspartic Acid