Protein Kinase C Activation Decreases Cell Surface Expression of the GLT-1 Subtype of Glutamate Transporter
2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês
10.1074/jbc.m203771200
ISSN1083-351X
AutoresAvtandil Kalandadze, Ying Wu, Michael B. Robinson,
Tópico(s)Receptor Mechanisms and Signaling
ResumoNa+-dependent glutamate transporters are required for the clearance of extracellular glutamate and influence both physiological and pathological effects of this excitatory amino acid. In the present study, the effects of a protein kinase C (PKC) activator on the cell surface expression and activity of the GLT-1 subtype of glutamate transporter were examined in two model systems, primary co-cultures of neurons and astrocytes that endogenously express GLT-1 and C6 glioma cells transfected with GLT-1. In both systems, activation of PKC with phorbol ester caused a decrease in GLT-1 cell surface expression. This effect is opposite to the one observed for the EAAC1 subtype of glutamate transporter (Davis, K. E., Straff, D. J., Weinstein, E. A., Bannerman, P. G., Correale, D. M., Rothstein, J. D., and Robinson, M. B. (1998) J. Neurosci. 18, 2475–2485). Several recombinant chimeric proteins between GLT-1 and EAAC1 transporter subtypes were generated to identify domains required for the subtype-specific redistribution of GLT-1. We identified a carboxyl-terminal domain consisting of 43 amino acids (amino acids 475–517) that is required for PKC-induced GLT-1 redistribution. Mutation of a non-conserved serine residue at position 486 partially attenuated but did not completely abolish the PKC-dependent redistribution of GLT-1. Although we observed a phorbol ester-dependent incorporation of 32P into immunoprecipitable GLT-1, mutation of serine 486 did not reduce this signal. We also found that chimeras containing the first 446 amino acids of GLT-1 were not functional unless amino acids 475–517 of GLT-1 were also present. These non-functional transporters were not as efficiently expressed on the cell surface and migrated to a smaller molecular weight, suggesting that a subtype-specific interaction is required for the formation of functional transporters. These studies demonstrate a novel effect of PKC on GLT-1 activity and define a unique carboxyl-terminal domain as an important determinant in cellular localization and regulation of GLT-1. Na+-dependent glutamate transporters are required for the clearance of extracellular glutamate and influence both physiological and pathological effects of this excitatory amino acid. In the present study, the effects of a protein kinase C (PKC) activator on the cell surface expression and activity of the GLT-1 subtype of glutamate transporter were examined in two model systems, primary co-cultures of neurons and astrocytes that endogenously express GLT-1 and C6 glioma cells transfected with GLT-1. In both systems, activation of PKC with phorbol ester caused a decrease in GLT-1 cell surface expression. This effect is opposite to the one observed for the EAAC1 subtype of glutamate transporter (Davis, K. E., Straff, D. J., Weinstein, E. A., Bannerman, P. G., Correale, D. M., Rothstein, J. D., and Robinson, M. B. (1998) J. Neurosci. 18, 2475–2485). Several recombinant chimeric proteins between GLT-1 and EAAC1 transporter subtypes were generated to identify domains required for the subtype-specific redistribution of GLT-1. We identified a carboxyl-terminal domain consisting of 43 amino acids (amino acids 475–517) that is required for PKC-induced GLT-1 redistribution. Mutation of a non-conserved serine residue at position 486 partially attenuated but did not completely abolish the PKC-dependent redistribution of GLT-1. Although we observed a phorbol ester-dependent incorporation of 32P into immunoprecipitable GLT-1, mutation of serine 486 did not reduce this signal. We also found that chimeras containing the first 446 amino acids of GLT-1 were not functional unless amino acids 475–517 of GLT-1 were also present. These non-functional transporters were not as efficiently expressed on the cell surface and migrated to a smaller molecular weight, suggesting that a subtype-specific interaction is required for the formation of functional transporters. These studies demonstrate a novel effect of PKC on GLT-1 activity and define a unique carboxyl-terminal domain as an important determinant in cellular localization and regulation of GLT-1. Glutamate is the predominant excitatory neurotransmitter in the central nervous system (CNS) 1The abbreviations used for: CNS, central nervous system; PKC, protein kinase C; DMEM, Dulbecco's modified Eagle's medium; GABA, γ-aminobutyric acid; HA, hemagglutinin; PMA, phorbol 12-myristate 13-acetate; Bis II, bisindolylmaleimide II; DHK, dihydrokainate. (1Fagg G.E. Foster A.C. Neuroscience (Oxford). 1983; 9: 701-719Crossref PubMed Scopus (563) Google Scholar, 2Fonnum F. J. Neurochem. 1984; 42: 1-11Crossref PubMed Scopus (1686) Google Scholar) and is removed from the synaptic cleft by sodium-dependent glutamate transport activity. This activity is mediated by a family of five subtypes of transporters that share up to 60% sequence identity (for reviews, see Refs. 3Amara S.G. Sonders M.S. Zahniser N.R. Povlock S.L. Daniels G.M. Adv. Pharmacol. 1998; 42: 164-168Crossref PubMed Scopus (39) Google Scholar, 4Sims K.D. Robinson M.B. Crit. Rev. Neurobiol. 1999; 13: 169-197Crossref PubMed Scopus (146) Google Scholar, 5Danbolt N.C. Prog. Neurobiol. 2001; 65: 1-105Crossref PubMed Scopus (3775) Google Scholar). Expression of these transporters is generally restricted to particular cell types and brain regions. Two subtypes, GLT-1 and GLAST, are astroglial and two others, EAAC1 and EAAT4, are neuronal. Expression of the fifth transporter, EAAT5, is restricted to the retina (for reviews, see Refs. 4Sims K.D. Robinson M.B. Crit. Rev. Neurobiol. 1999; 13: 169-197Crossref PubMed Scopus (146) Google Scholar and 5Danbolt N.C. Prog. Neurobiol. 2001; 65: 1-105Crossref PubMed Scopus (3775) Google Scholar). Both the neuronal and glial glutamate transporters control the amplitude and/or duration of synaptic responses (6Mennerick S. Zorumski C.F. Nature. 1994; 368: 59-62Crossref PubMed Scopus (291) Google Scholar, 7Otis T.S. Kavanaugh M.P. Jahr C.E. Science. 1997; 277: 1515-1518Crossref PubMed Scopus (156) Google Scholar) and prevent an extracellular accumulation of this potential excitotoxin (8Robinson M.B. Djali S. Buchhalter J.R. J. Neurochem. 1993; 61: 2099-2103Crossref PubMed Scopus (100) Google Scholar, 9Rothstein J.D. Dykes-Hoberg M. Pardo C.A. Bristol L.A. Jin L. Kuncl R.W. Kanai Y. Hediger M. Wang Y. Schielke J.P. Welty D.F. Neuron. 1996; 16: 675-686Abstract Full Text Full Text PDF PubMed Scopus (2142) Google Scholar, 10Tanaka K. Watase K. Manabe T. Yamada K. Watanabe M. Takahashi K. Iwama H. Nishikawa T. Ichihara N. Kikuchi T. Okuyama S. Kawashima N. Hori S. Takimoto M. Wada K. Science. 1997; 276: 1699-1702Crossref PubMed Scopus (1481) Google Scholar). For several different reasons, it is thought that the glial transporter, GLT-1, may represent the predominant route for the clearance of extracellular glutamate in forebrain (for review, see Ref.11Robinson M.B. Neurochem. Int. 1999; 33: 479-491Crossref Scopus (21) Google Scholar). Therefore, defining the mechanisms that regulate GLT-1 has the potential to impact our understanding of both the physiology and pathology of glutamate in the CNS. Several different second messengers regulate the activity of GLT-1, including free radicals, arachidonic acid, and PKC (for reviews, see Refs. 4Sims K.D. Robinson M.B. Crit. Rev. Neurobiol. 1999; 13: 169-197Crossref PubMed Scopus (146) Google Scholar and 5Danbolt N.C. Prog. Neurobiol. 2001; 65: 1-105Crossref PubMed Scopus (3775) Google Scholar). However, the effects of PKC on GLT-1-mediated activity are varied. For example, activation of PKC causes an increase in activity when GLT-1 is expressed in HeLa cells using vaccinia virus (12Casado M. Bendahan A. Zafra F. Danbolt N.C. Gimenez C. Kanner B.I. J. Biol. Chem. 1993; 268: 27313-27317Abstract Full Text PDF PubMed Google Scholar) but has no effect on the activity of GLT-1 stably transfected into HeLa cells or two other peripheral cell lines (13Tan J. Zelenaia O. Rothstein J.D. Robinson M.B. J. Pharmacol. Exp. Ther. 1999; 289: 1600-1610PubMed Google Scholar). In a cell line that endogenously expresses the human variant of GLT-1 (Y-79 human retinoblastoma), activation of PKC causes a decrease in activity by increasing the K m value for transport (14Ganel R. Crosson C.E. J. Neurochem. 1998; 70: 993-1000Crossref PubMed Scopus (49) Google Scholar). In a preliminary study, activation of PKC caused a decrease in GLT-1 activity in stably transfected Madin-Darby canine kidney cells (15Carrick T. Dunlop J. Soc. Neurosci. Abstr. 1999; 25: 426Google Scholar). These studies suggest that the effects of PKC activation may be dependent on the levels of transporter expression and/or the presence of other cellular proteins that are not constitutively expressed in all cells. Many types of membrane-bound proteins undergo a dynamic trafficking between the cell surface and intracellular compartments. Changes in cell surface expression can up- or down-regulate the activity of membrane proteins much faster (within minutes) than can normally be achieved by altering the rate of protein synthesis. One well-characterized example is up-regulation of the GLUT4 subtype of glucose transporter, which is rapidly redistributed to the plasma membrane in response to insulin (for reviews, see Refs. 16Czech M.P. Corvera S. J. Biol. Chem. 1999; 274: 1865-1868Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar and 17Pessin J.E. Thurmond D.C. Elmendorf J.S. Coker K.J. Okada S. J. Biol. Chem. 1999; 274: 2593-2596Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). A classic example of down-regulation is agonist-activated internalization of G-protein-coupled receptors (for review, see Ref. 18Ferguson S.S.G. Pharmacol. Rev. 2001; 53: 1-24PubMed Google Scholar). Several groups have recently shown that activation of PKC causes a rapid change in the cell surface expression of neurotransmitter transporters, including the GAT1 subtype of GABA transporter (19Beckman M.L. Bernstein E.M. Quick M.W. J. Neurosci. 1999; 19: 1-6PubMed Google Scholar), the dopamine transporter (20Daniels G.M. Amara S.G. J. Biol. Chem. 1999; 274: 35794-35801Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 21Melikian H.E. Buckley K.M. J. Neurosci. 1999; 19: 7699-7710Crossref PubMed Google Scholar), and the serotonin transporter (22Qian Y. Galli A. Ramamoorthy S. Risso S. DeFelice L.J. Blakely R.D. J. Neurosci. 1997; 17: 45-57Crossref PubMed Google Scholar). In view of these observations, it may not be unexpected that trafficking of glutamate transporters is also controlled. In fact, our previous studies indicate that the cell surface expression of the neuronal glutamate transporter, EAAC1, is increased by activating PKC or by activating the platelet-derived growth factor receptor (23Sims K.D. Straff D.J. Robinson M.B. J. Biol. Chem. 2000; 274: 5228-5327Abstract Full Text Full Text PDF Scopus (116) Google Scholar, 24Davis K.E. Straff D.J. Weinstein E.A. Bannerman P.G. Correale D.M. Rothstein J.D. Robinson M.B. J. Neurosci. 1998; 18: 2475-2485Crossref PubMed Google Scholar). In the present study, the effects of a PKC activator on GLT-1 cell surface expression were examined in co-cultures of neurons and astrocytes. In this model system, phorbol ester caused a rapid decrease in the cell surface expression of GLT-1. When GLT-1 was introduced into C6 glioma cells, a CNS-derived cell line that endogenously expresses the neuronal transporter, EAAC1, a PKC activator caused a decrease in the cell surface expression of GLT-1 and a decrease in GLT-1-mediated transport activity. In contrast, a PKC activator caused an increase in EAAC1 cell surface expression in these same experiments, indicating that the differential effects of PKC are related to differences in primary structure of these two highly homologous proteins. Using chimeras made of reciprocal domains of GLT-1 and EAAC1, we demonstrate that a 43-amino acid carboxyl-terminal domain is both necessary and sufficient for PKC-mediated redistribution of GLT-1. Following mutation of serine and threonine residues in this domain, we show that a non-conserved serine residue (serine 486) is partially responsible for the regulated decrease in GLT-1 cell surface expression. Although we found that a PKC activator increased incorporation of 32P into wild type GLT-1, mutation of this single serine residue did not reduce this signal. During these studies, we also developed evidence that the same 43-mino acid domain of GLT-1 was required for functional expression of transporter activity in specific chimeras. Together, these studies provide evidence for PKC-dependent regulation of GLT-1 and identify a structural domain required for this effect. DMEM, l-glutamine, and penicillin/streptomycin were obtained from Invitrogen (Gaithersburg, MD). Fetal bovine serum was purchased from HyClone (Logan, UT). Sulfosuccinimidobiotin and Immunopure immobilized monomeric avidin were from Pierce (Rockford, IL). Donkey anti-rabbit horseradish peroxidase IgG, rainbow molecular weight markers, and enhanced chemiluminescence kits were purchased from Amersham Biosciences (Arlington Heights, IL). Immobilon P (polyvinylidene fluoride membrane) was from Millipore (Bedford, MA).l-[3H]Glutamate was obtained from PerkinElmer Life Sciences (Boston, MA); [32P]H3PO4 was purchased from ICN (Irvine, CA). Dihydrokainate was purchased from Tocris (Ballwin, MO). Polyclonal anti-GLT-1 and anti-EAAC1 antibodies, raised against peptide sequences from carboxyl termini of these transporters, were given generously by Dr. Rothstein (25Rothstein J.D. Martin L. Levey A.I. Dykes-Hoberg M. Jin L. Wu D. Nash N. Kuncl R.W. Neuron. 1994; 13: 713-725Abstract Full Text PDF PubMed Scopus (1462) Google Scholar). Mouse monoclonal anti-hemagglutinin (anti-HA) antibody was obtained from Roche Molecular Biochemicals (Indianapolis, IN). Bisindolylmaleimide II (Bis II) was obtained fromCalbiochem (La Jolla, CA). GenePorter transfection reagent was purchased from Gene therapy Systems (San Diego, CA). Anti-actin antibody, phorbol 12-myristate 13-acetate (PMA), and poly-d-lysine were purchased from Sigma (St. Louis, MO). Neuron/astrocyte-mixed cultures were prepared from embryonic day 17–19 cortices of rat as described previously (8Robinson M.B. Djali S. Buchhalter J.R. J. Neurochem. 1993; 61: 2099-2103Crossref PubMed Scopus (100) Google Scholar) with minor modifications. Cells were plated onto poly-d-lysine (50 μg/ml)-coated plastic dishes and maintained in a 7% CO2 incubator at 37 °C. The cultures were fed with a one-third medium exchange twice a week. After 7–10 days, neurons in these cultures sit on top of a monolayer of astrocytes and represent 3 mm) (Refs. 13Tan J. Zelenaia O. Rothstein J.D. Robinson M.B. J. Pharmacol. Exp. Ther. 1999; 289: 1600-1610PubMed Google Scholar,35Arriza J.L. Fairman W.A. Wadiche J.I. Murdoch G.H. Kavanaugh M.P. Amara S.G. J. Neurosci. 1994; 14: 5559-5569Crossref PubMed Google Scholar, and 36Dowd L.A. Coyle A.J. Rothstein J.D. Pritchett D.B. Robinson M.B. Mol. Pharmacol. 1996; 49: 465-473PubMed Google Scholar, for review, see Ref. 37Robinson M.B. Dowd L.A. Adv. Pharmacol. 1997; 37: 69-115Crossref PubMed Scopus (181) Google Scholar). Therefore, low concentrations of DHK should selectively inhibit GLT-1-mediated activity. In initial studies, the effects of increasing concentrations of DHK on activity were examined in stably transfected and untransfected C6 glioma. In these experiments, inhibition of transport activity by DHK was consistent with a single population of sites with an IC50value of 1100 μm (n = 2). In C6-GLT-1 cells, the data for inhibition of transport activity were best fit to two sites with IC50 values of 16 and 1300 μm; 31% of the sites were of higher affinity (n = 2). This percentage of high affinity sites (GLT-1-mediated activity) is consistent with the higher V max value observed in these cells compared with controls (see previous paragraph). This C6-GLT-1 clone was used to determine if PMA reduces the level of DHK-sensitive transport. The effects of PMA on total transporter activity and DHK-sensitive activity were compared in untransfected and stably transfected cell lines. In untransfected cells, PMA increased Na+-dependentl-[3H]-Glu transport activity, and 100 μm DHK had essentially no effect on activity in both vehicle and PMA-treated cells (Fig. 3 C). In C6-GLT-1 cells, PMA had no effect on total transporter activity, but PMA decreased DHK-sensitive transport activity (Fig. 3 D). The reduction in DHK-sensitive transport activity, to ∼40% of control, is in close agreement with the reduction in GLT-1 cell surface expression observed in this cell line (Fig. 3 B). These data suggest that PMA decreases GLT-1-mediated activity and demonstrate that this decrease in activity is asso
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