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Cyclic AMP and Synaptic Activity-Dependent Phosphorylation of AMPA-Preferring Glutamate Receptors

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Cyclic AMP and Synaptic Activity-Dependent Phosphorylation of AMPA-Preferring Glutamate Receptors The Journal of Neuroscience, December 1994, 1412): 7585-7593 Cyclic AMP and Synaptic Activity-Dependent Phosphorylation of AMPA-Preferring Glutamate Receptors Craig Blackstone,lva Timothy H. Murphy,lrb Stephen J. Moss,~~~~~ Jay M. Baraban,lm* and Richard L. Huganir1v3 ‘Departments of Neuroscience, ‘Psychiatry and Behavioral Sciences, and the 3Howard Hughes Medical Institute, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Several studies have suggested that the function of gluta- protein kinase C. These results indicate that AMPA receptors mate receptor channels can be regulated by protein phos- containing the GIuRl subunit may be regulated by extra- phorylation. Furthermore, a basal level of phosphorylation cellular signals working through the CAMP second messen- may be necessary to maintain receptor function. Little is ger system as well as by synaptic activity, possibly acting known, however, about the phosphorylation state of gluta- through protein kinase C. Such regulation by protein phos- mate receptor channels in neurons and how it is regulated phorylation may be involved in short-term changes in syn- by synaptic activity. In this study, we have investigated the aptic efficacy thought to involve the functional modulation phosphorylation of the AMPA-preferring glutamate receptor of AMPA receptors. subunit GluRl in cortical neurons in primary culture. These [Key words: glutamate, AMPA receptor, phosphorylation, neurons elaborate extensive processes, form functional syn- CAMP, synaptic activity, long-term potentiation] apses, and exhibit spontaneous 4-8 set bursts of synaptic activity every 15-20 sec. In cultures in which this synaptic Glutamate receptors mediate excitatory neurotransmission in activity was suppressed by tetrodotoxin and MK-801, the the CNS, playing critical roles during synaptogenesis,in mech- GIuRl protein was phosphorylated on serine residues within anismsof synaptic plasticity suchas long-term potentiation (LTP) a single tryptic phosphopeptide, as determined by phos- and long-term depression(LTD), and in someneuropathological phoamino acid analysis and phosphopeptide mapping. This conditions (Ito et al., 1989; Siegelbaumand Kandel, 1991; Bliss same peptide was basally phosphorylated in recombinant and Collingridge, 1993; Shaw, 1993; Linden, 1994; Lipton and GluRl receptors transiently expressed in human embryonal Rosenberg,1994). Thesereceptors can be divided into two broad kidney 293 cells. Treatment of these synaptically inactive categories: ionotropic receptors, oligomeric proteins that form cortical neurons with the adenylyl cyclase activator forskolin ligand-gated ion channels; and metabotropic receptors, mono- resulted in a robust increase in phosphorylation on serine mers that are coupled through guanine nucleotide-binding pro- residues on a phosphopeptide distinct from the basally teins to second messengercascades. The ionotropic receptors phosphorylated peptide. Again, this same phosphopeptide have classically been divided into a-amino-3-hydroxy-s-meth- was observed in recombinant GluRl receptors isolated from yl-4-isoxazolepropionate (AMPA), kainate, and N-methyl-D-as- 293 cells coexpressing the catalytic subunit of CAMP-de- partate (NMDA) receptors basedon their preferred agonistsand pendent protein kinase. Spontaneous synaptic activity in electrophysiological properties (Monaghan et al., 1989). Over cultures of cortical neurons resulted in a consistent, rapid the past several years, molecular cloning studieshave identified (within 1 O-30 set) increase in phosphorylation on serine and many subunits of the ionotropic receptors. These have been threonine residues. Interestingly, these phosphopeptides classifiedon the basisof sequencesimilarity and pharmacology were also phosphorylated when neurons from inactive cul- as membersof the AMPA (GluR1-4) kainate (GluR5-7, KAl, tures were stimulated with phorbol esters, which activate 2), and NMDA (NMDAR 1, 2A-D) receptor complexes (Gasic and Hollmann, 1992; Nakanishi, 1992; Sommer and Seeburg, 1992; Seeburg, 1993; Hollmann and Heinemann, 1994). Two Received Oct. 11, 1993; revised May 20, 1994; accepted June 2, 1994. additional subunits related in sequence(6 1 and 62) remain “or- C.B. and T.H.M. contributed equally to this study. We are grateful to Drs. Michael Hollmann and Stephen Heinemann for the GluR 1 RoPcDNA and to Dr. phan” receptors (Yamazaki et al., 1992; Lomeli et al., 1993). Michael D. Uhler for the cDNA of the catalytic subunit of PKA (Ca). This work Further diversity is generatedthrough alternative RNA splicing was supported by an MSTP fellowship (C.B.), an NRSA fellowship (T.H.M.), USPHS Grants DA-00266 and MH-00926 (J.M.B.), and the Howard Hughes and posttranscriptional mRNA editing of many of these sub- Medical Institute (R.L.H.). units (Sommer et al., 1990, 1991; Gallo et al., 1992; Sugihara Correspondence should be addressed to Dr. Richard L. Huganir, Department et al., 1992; Seeburg, 1993).The predicted amino acid sequences of Neuroscience, Howard Hughes Medical Institute, The Johns Hopkins Univer- sity School of Medicine, 725 North Wolfe Street, PCTB 900, Baltimore, MD derived from these cloning studies have permitted the genera- 21205-2185. tion of subunit-specific antibodies against these proteins, facil- 1 Present address: Harvard-Longwood Neurology Program and Harvard Med- itating the study of such biochemical properties asreceptor sub- ical School, Boston, MA 02 115. b Present address: Kinsmen Laboratory for Neurological Research, Division of unit composition and modification by protein phosphorylation Neurological Sciences, Department of Psychiatry, University of British Columbia, and glycosylation (Rogerset al., 1991; Blackstone et al., 1992b; Vancouver, BC V6T 123, Canada. c Present address: IRC Institute for Molecular Cell Biology and Department of Wenthold et al., 1992, 1994; Moss et al., 1993; Raymond et al., Pharmacology, University College, London WClE 6BT, UK. 1993b; Roche et al., 1994; Sheng et al., 1994). Copyright 0 1994 Society for Neuroscience 0270-6474/94/147585-09$05.00/O One posttranslational mechanismimplicated in the long-term 7586 Blackstone et al. - Glutamate Receptor Phosphorylation functional modulation of glutamate receptors is phosphoryla- rnM NaCl, 5.0 mM KCl, 2.5 mM CaCl,, 1.0 mM MgSO,, 10 mM sodium tion by intracellular protein kinases (Blackstone et al., 1992~; HEPES, 1 mM NaHCO,, and 22 mM glucose (pH 7.4) supplemented with l-2 mCi/ml 32P- orthophosphate. The labeling medium was then Swope et al., 1992; Raymond et al., 1993b). Application of removed and replaced with phosphate-free HBSS containing picrotoxin activators of CAMP-dependent protein kinase (PKA) as well as ( 10 PM) for synaptically active cultures, or picrotoxin ( 10 PM), TTX ( 1 purified PKA has been shown to potentiate the function of PM), and MK-80 1 (3 PM) for synaptically inactive cultures. In the pres- AMPA/kainate receptors in cultured rat hippocampal neurons ence of the GABA, receptorchannel antagonist picrotoxin, virtually all (Greengard et al., 1991; Wang et al., 1991) and teleost retinal cortical neurons within a culture exhibit highly synchronous sponta- neous synaptic activity; a combination of TTX and MK-80 1 is used to horizontal cells (Liman et al., 1989). CAMP analogs, possibly block synaptic activity in these cultures (Murphy et al., 1992a, 1994). working through PKA, increase the current and calcium influx In experiments utilizing activators of intracellular protein kinases, the through recombinant GluRl/GluR3 AMPA receptor channels “synaptically inactive” culture solution was supplemented with either expressed in Xenopus oocytes (Keller et al., 1992). Similarly, forskolin (10 PM) and IBMX (75 PM), or PDA (5 PM) whereindicated. Cultures were incubated with these solutions for 20 min at room tem- homomeric GluR6 kainate receptors transiently expressed in perature unless otherwise indicated. human embryonal kidney 293 cells are directly phosphorylated We sought to determine the time course of GluRl phosphorylation and potentiated by PKA (Raymond et al., 1993a; Wang et al., after the initiation of synaptic activity. Since these cultures show prom- 1993). Another serine/threonine kinase, calcium/calmodulin- inent spontaneous firing and associated protein kinase activity (Murphy dependent protein kinase II (CaMKII), also enhances kainate- et al., 1994) we needed a method to suppress the endogenous synaptic activity that was readily reversible. Previous observations indicated that gated currents in cultured rat hippocampal neurons (McGlade- lowering extracellular calcium to 0.5 mM and raising magnesium to 2 McCulloh et al., 1993). These reports strongly support a key mM suppressed the amplitude and duration of intracellular calcium role for direct protein phosphorylation of AMPA/kainate glu- transients, without affecting resting intracellular calcium levels (Murphy tamate receptors in altering the excitability of neurons in the et al., 1992a, 1994). Cortical cultures were labeled with l-2 mCi/ml 32P-orthophosphate for 4 hr in a medium containing phosphate-free CNS. HBSS with low calcium (0.5 mM vs 2.25 mM), high magnesium (2 mM Previous studies have shown that the recombinant GluRl,,, vs 1 mM), and picrotoxin (10 PM). Restoration of normal calcium and (Hollmann et al., 1989) AMPA receptor protein expressed in magnesium concentrations (2.25 mM and 1 mM, respectively) by adding 293 cells is a substrate for both tyrosine and serine/threonine an equal volume of medium containing 4 mM calcium
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