title: Differential regulation of two isoforms of the glial glutamate transporter EAAT2 by DLG1 and CaMKII.
abstract: The gene for EAAT2, the major astrocytic glutamate transporter, generates two carrier isoforms (EAAT2a and EAAT2b) that vary at their C termini as a consequence of alternative RNA splicing. The EAAT2b cytoplasmic C terminus contains a postsynaptic density-95/Discs large/zona occludens-1 (PDZ) ligand, which is absent in EAAT2a. To understand how the distinct C termini might affect transporter trafficking and surface localization, we generated Madin-Darby canine kidney (MDCK) cells that stably express EGFP-EAAT2a or EGFP-EAAT2b and found robust basolateral membrane expression of the EAAT2b isoform. In contrast, EAAT2a displayed a predominant distribution within intracellular vesicle compartments, constitutively cycling to and from the membrane. Addition of the PDZ ligand to EAAT2a as well as its deletion from EAAT2b confirmed the importance of the motif for cell-surface localization. Using EAAT2 constructs with an extracellular biotin acceptor tag to directly assess surface proteins, we observed significant PDZ ligand-dependent EAAT2b surface expression in cultured astrocytes, consistent with observations in cell lines. Discs large homolog 1 (DLG1; SAP97), a PDZ protein prominent in both astrocytes and MDCK cells, colocalized and coimmunoprecipitated with EAAT2b. shRNA knockdown of DLG1 expression decreased surface EAAT2b in both MDCK cells and cultured astrocytes, suggesting that the DLG scaffolding protein stabilizes EAAT2b at the surface. DLG1 can be phosphorylated by Ca(2+)/calmodulin-dependent protein kinase (CaMKII), resulting in disruption of its PDZ-mediated interaction. In murine astrocytes and acute brain slices, activation of CaMKII decreases EAAT2b surface expression but does not alter the distribution of EAAT2a. These data indicate that the surface expression and function of EAAT2b can be rapidly modulated through the disruption of its interaction with DLG1 by CaMKII activation.
authors.affiliation: Laboratory of Molecular and Cellular Neuroscience, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892 and suzanne.underhill@nih.gov.
authors.forename: Suzanne M
authors.initials: SM
authors.lastname: Underhill
authors.affiliation: Department of Cellular Biology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15260.
authors.forename: David S
authors.identifier: http://orcid.org/0000-0001-9355-144X
authors.initials: DS
authors.lastname: Wheeler
authors.affiliation: Laboratory of Molecular and Cellular Neuroscience, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892 and.
authors.forename: Susan G
authors.initials: SG
authors.lastname: Amara
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D000074288:Discs Large Homolog 1 Protein; D004285:Dogs; D027342:Excitatory Amino Acid Transporter 2 / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D051379:Mice; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D054731:PDZ Domains / Q000235:genetics; D020033:Protein Isoforms / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D000074407:SAP90-PSD95 Associated Proteins
chemical_list: D000074288:Discs Large Homolog 1 Protein; C000614189:Dlg1 protein, mouse; D027342:Excitatory Amino Acid Transporter 2; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D000074407:SAP90-PSD95 Associated Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2
keywords: CaMKII; DLG1; EAAT2; PDZ; glutamate transporter
references.citation: J Comp Neurol. 2000 Jan 3;416(1):6-18
references.pmid: 10578099
references.citation: Nat Neurosci. 2015 Feb;18(2):219-26
references.pmid: 25581361
references.citation: Prog Neurobiol. 2001 Sep;65(1):1-105
references.pmid: 11369436
references.citation: Mol Biol Rep. 2000;27(4):217-24
references.pmid: 11455957
references.citation: Prog Brain Res. 2001;132:287-99
references.pmid: 11544996
references.citation: Neuroscience. 2002;109(1):45-61
references.pmid: 11784699
references.citation: J Neurosci. 2002 Mar 15;22(6):2142-52
references.pmid: 11896154
references.citation: J Comp Neurol. 2002 Jun 10;447(4):323-30
references.pmid: 11992519
references.citation: J Biol Chem. 2002 Jun 14;277(24):21697-711
references.pmid: 11937501
references.citation: Ann Med. 2002;34(2):109-19
references.pmid: 12108574
references.citation: J Neurosci. 2002 Dec 15;22(24):10643-52
references.pmid: 12486157
references.citation: J Neurosci. 2003 Jun 1;23(11):4567-76
references.pmid: 12805297
references.citation: J Biol Chem. 2003 Nov 7;278(45):44745-52
references.pmid: 12933808
references.citation: Biochemistry. 2003 Nov 11;42(44):12981-8
references.pmid: 14596613
references.citation: Glia. 2004 Jan 15;45(2):155-69
references.pmid: 14730709
references.citation: Neurobiol Dis. 2004 Apr;15(3):461-73
references.pmid: 15056453
references.citation: J Neurosci. 2004 May 12;24(19):4551-9
references.pmid: 15140926
references.citation: J Biol Chem. 2004 May 28;279(22):23813-21
references.pmid: 15044483
references.citation: Curr Opin Neurobiol. 2004 Jun;14(3):346-52
references.pmid: 15194115
references.citation: J Biol Chem. 2004 Sep 17;279(38):39505-12
references.pmid: 15265858
references.citation: Nature. 2004 Oct 14;431(7010):811-8
references.pmid: 15483603
references.citation: Ann Neurol. 1986 Feb;19(2):105-11
references.pmid: 2421636
references.citation: J Biol Chem. 1994 Feb 18;269(7):5428-39
references.pmid: 7906275
references.citation: Mol Pharmacol. 1995 Oct;48(4):648-57
references.pmid: 7476890
references.citation: FEBS Lett. 1997 Oct 27;416(3):312-6
references.pmid: 9373176
references.citation: J Biol Chem. 1998 Jul 31;273(31):19518-24
references.pmid: 9677374
references.citation: Glia. 2005 Jan 1;49(1):121-33
references.pmid: 15390098
references.citation: J Cell Biol. 2005 Sep 12;170(6):895-901
references.pmid: 16157700
references.citation: Neuron. 2006 Jul 6;51(1):99-111
references.pmid: 16815335
references.citation: J Biol Chem. 2007 Sep 28;282(39):28691-9
references.pmid: 17635915
references.citation: J Biol Chem. 2007 Dec 7;282(49):35564-73
references.pmid: 17913704
references.citation: Eur J Neurosci. 2008 Jan;27(1):66-82
references.pmid: 18184314
references.citation: Nat Protoc. 2008;3(3):534-45
references.pmid: 18323822
references.citation: J Neurochem. 2008 Jun;105(5):1834-48
references.pmid: 18248606
references.citation: Mol Pharmacol. 2009 May;75(5):1062-73
references.pmid: 19201818
references.citation: J Neurochem. 2009 Jul;110(1):264-74
references.pmid: 19457061
references.citation: Neuroscience. 2009 Sep 15;162(4):1055-71
references.pmid: 19328838
references.citation: Ethn Dis. 2010 Winter;20(1 Suppl 1):S1-28-32
references.pmid: 20521381
references.citation: J Biol Chem. 2010 Oct 8;285(41):31313-24
references.pmid: 20688910
references.citation: J Biol Chem. 2011 Feb 4;286(5):3935-43
references.pmid: 21127051
references.citation: Proc Natl Acad Sci U S A. 2011 Sep 13;108(37):15141-6
references.pmid: 21876140
references.citation: Neurochem Int. 2013 Aug;63(2):61-8
references.pmid: 23665075
references.citation: J Comp Neurol. 2014 Jan 1;522(1):204-24
references.pmid: 23939750
references.citation: Neuron. 2014 Jul 16;83(2):404-16
references.pmid: 25033183
references.citation: Oncogene. 1999 Dec 16;18(54):7810-5
references.pmid: 10618722