Inhibitory Interactions between Phosphorylation Sites in the C Terminus of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid-type Glutamate Receptor GluA1 Subunits

Gray, Erin E.; Guglietta, Ryan; Khakh, Baljit S.; O’Dell, Thomas J.

doi:10.1074/jbc.m114.553537

Cited by 11 publications

(18 citation statements)

References 53 publications

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“…Email: rhuganir@jhmi.edu. pS831, pS845, and pT840 antibodies (10,16,17). Because the GluA1 pT840 antibody detected several bands, we confirmed the specificity of our antibody using the GluA1 "penta" knock-in mouse, which harbors mutations at GluA1 S831A, T838A, S839A, T840A, and S845A (15).…”

Section: Resultssupporting

confidence: 62%

Regulation of AMPA receptor phosphorylation by the neuropeptide PACAP38

Toda

Huganir

2015

Proc. Natl. Acad. Sci. U.S.A.

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Dynamic changes in synaptic strength are thought to be critical for higher brain function such as learning and memory. Alterations in synaptic strength can result from modulation of AMPA receptor (AMPAR) function and trafficking to synaptic sites. The phosphorylation state of AMPAR subunits is one mechanism by which cells regulate receptor function and trafficking. Receptor phosphorylation is in turn regulated by extracellular signals; these include neuronal activity, neuropeptides, and neuromodulators such as dopamine and norepinephrine (NE). Although numerous studies have reported that the neuropeptide pituitary adenylate cyclase activating polypeptide 38 (PACAP38) alters hippocampal CA1 synaptic strength and GluA1 synaptic localization, its effect on AMPAR phosphorylation state has not been explored. We determined that PACAP38 stimulation of hippocampal cultures increased phosphorylation of S845, and decreased phosphorylation of T840 on the GluA1 AMPAR subunit. Increases in GluA1 S845 phosphorylation primarily occurred via PAC1 and VPAC2 receptor activation, whereas a reduction in GluA1 T840 phosphorylation was largely driven by PAC1 receptor activation and to a lesser extent by VPAC1 and VPAC2 receptor activation. GluA1 S845 phosphorylation could be blocked by a PKA inhibitor, and GluA1 T840 dephosphorylation could be blocked by a protein phosphatase 1/2A (PP1/PP2A) inhibitor and was partly blocked by a NMDA receptor (NMDAR) antagonist. These results demonstrate that the neuropeptide PACAP38 inversely regulates the phosphorylation of two distinct sites on GluA1 and may play an important role modulating AMPAR function and synaptic plasticity in the brain.PACAP38 | AMPA receptor phosphorylation | synaptic transmission A MPA-type glutamate receptors (AMPARs) are a tetrameric assembly composed of the GluA1, 2, 3, or 4 subunits. Within the adult hippocampus, receptors consist of primarily GluA1/2 and GluA2/3 complexes (1). Because AMPARs conduct the majority of excitatory transmission in the brain, modulation of AMPAR synaptic transmission is a powerful tool by which the cell can regulate synaptic strength and cell firing. Furthermore, it is hypothesized that complex behaviors such as learning, memory, and drug addiction involve alterations in synaptic strength (2, 3).The cell can regulate synaptic strength through changes in AMPAR conductance, trafficking, and tethering at synaptic sites. Such changes can be achieved through alterations in AMPAR expression, binding partners, and posttranslational modifications (4). A number of GluA1 and GluA2 phosphorylation sites have been proposed to play a role in AMPAR trafficking and synaptic plasticity. GluA1 S845 and T840 are two phosphorylation sites particularly relevant to this study. GluA1 S845 is phosphorylated by PKA and cGMP-dependent protein kinase II (5, 6). Its phosphorylation levels are regulated by NMDA receptors (NMDARs) (7), β-adrenergic receptors (8, 9), and muscarinic cholinergic receptors (9), and during homeostatic scaling (10), long-term depression (LTD...

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Section: Resultssupporting

confidence: 62%

Regulation of AMPA receptor phosphorylation by the neuropeptide PACAP38

Toda

Huganir

2015

Proc. Natl. Acad. Sci. U.S.A.

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“…It is important to note, however, that our use of crude hippocampal homogenates makes it impossible to rule out the possibility that different proportions of S845-phosphorylated GluA1 exist in different cellular compartments, cell types, and hippocampal regions. However, our observations (and those of Hosokawa et al 2015) do help to explain the paradoxical finding that under some experimental conditions pharmacological activation of NMDARs can induce robust and persistent decreases in S845 phosphorylation (as detected by immunoblotting with phospho-specific antibodies) yet have no lasting effect on synaptic strength (Gray et al 2014). In contrast, the induction of shortterm depression of synaptic transmission by bath application of NMDA induces a transient dephosphorylation of GluA1 at T840 that recovers with a time course similar to the recovery of synaptic transmission.…”

Section: Glua1 Phosphorylation In Hippocampal Neuronsmentioning

confidence: 73%

“…In contrast, the induction of shortterm depression of synaptic transmission by bath application of NMDA induces a transient dephosphorylation of GluA1 at T840 that recovers with a time course similar to the recovery of synaptic transmission. Furthermore, this site is persistently dephosphorylated following the induction of LTD by a chemical induction protocol (Delgado et al 2007;Gray et al 2014). Thus, unlike S845, dephosphorylation of T840 closely parallels changes in synaptic strength during both short-term and long-term decreases in synaptic strength.…”

Section: Glua1 Phosphorylation In Hippocampal Neuronsmentioning

confidence: 87%

“…Horseradish peroxidase conjugated secondary antibodies (1:2,000) were obtained from GE Healthcare. The phospho-T840 and phospho-S845 GluA1 antibodies used in these experiments have been extensively characterized by us (Delgado et al 2007;Gray et al 2014) and others (Hosokawa et al 2015;Toda and Huganir 2015) and do not recognize nonphosphorylated GluA1 or GluA1 phosphorylated at other sites.…”

Section: Reagents and Antibodiesmentioning

confidence: 99%

“…Moreover, LTD is disrupted in mutant mice where S845 is replaced with a nonphosphorylatable alanine (Lee et al 2003(Lee et al , 2010He et al 2011). Phosphorylation of GluA1 subunits at a nearby site, threonine 840 (T840), enhances the conductance of AMPAR ion channels (Jenkins et al 2014), and this site is also dephosphorylated in LTD (Delgado et al 2007;Babiec et al 2014;Gray et al 2014). Thus, dephosphorylation of T840 may also have a role in LTD. Interestingly, and somewhat paradoxically, LTD can still be induced in CA1 pyramidal cells expressing AMPA receptor GluA1 subunits that lack the entire carboxy-terminus (Granger and Nicoll 2014) and in hippocampal slices from GluA1 null mutant mice (Selcher et al 2012).…”

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confidence: 99%

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Basal levels of AMPA receptor GluA1 subunit phosphorylation at threonine 840 and serine 845 in hippocampal neurons

2016

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Dephosphorylation of AMPA receptor (AMPAR) GluA1 subunits at two sites, serine 845 (S845) and threonine 840 (T840), is thought to be involved in NMDA receptor-dependent forms of long-term depression (LTD). Importantly, the notion that dephosphorylation of these sites contributes to LTD assumes that a significant fraction of GluA1 subunits are basally phosphorylated at these sites. To examine this question, we used immunoprecipitation/depletion assays to estimate the proportion of GluA1 subunits basally phosphorylated at S845 and T840. Although dephosphorylation of S845 is thought to have a key role in LTD, our results indicate that few GluA1 subunits in hippocampal neurons are phosphorylated at this site. In contrast, 50% of GluA1 subunits are basally phosphorylated at T840, suggesting that dephosphorylation of this site can contribute to the down-regulation of AMPAR-mediated synaptic transmission in LTD.

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Dynamic Regulation of AMPAR Phosphorylation In Vivo Following Acute Behavioral Stress

et al. 2016

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The tuning of glutamatergic transmission is an essential mechanism for neuronal communication. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ionotropic glutamate receptors that mediate fast synaptic transmission. The phosphorylation states of specific serine residues on the GluA1 and GluA2 AMPAR subunits are considered critical post-translational modifications that regulate AMPAR activity and subcellular trafficking. While behavioral stress, via stress hormones, exerts specific alterations on such glutamatergic processes, there have been conflicting data concerning the influence of stress on AMPAR phosphorylation in different brain regions, and the post-stress signaling mechanisms mediating these processes are not well delineated. Here, we examined the dynamics of phosphorylation at three AMPAR serine residues (ser831-GluA1, ser845-GluA1, and ser880-GluA2) in four brain regions [amygdala, medial prefrontal cortex (mPFC), dorsal hippocampus, and ventral hippocampus] of the rat during the hour following behavioral stress. We also tested the impact of post-stress corticosteroid receptor blockade on AMPAR phosphorylation. Both GluA1 subunit residues exhibited elevated phosphorylation after stress, yet post-stress administration of corticosteroid receptor antagonists curtailed these effects only at ser831-GluA1. In contrast, ser880-GluA2 displayed a time-dependent tendency for early decreased phosphorylation (that was selectively augmented by mifepristone treatment in the amygdala and mPFC of stressed animals) followed by increased phosphorylation later on. These findings show that the in vivo regulation of AMPAR phosphorylation after stress is a dynamic and subunit-specific process, and they provide support for the hypothesis that corticosteroid receptors have an ongoing role in the regulation of ser831-GluA1 phosphorylation during the post-stress interval.

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Inhibitory Interactions between Phosphorylation Sites in the C Terminus of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid-type Glutamate Receptor GluA1 Subunits

Cited by 11 publications

References 53 publications

Regulation of AMPA receptor phosphorylation by the neuropeptide PACAP38

Regulation of AMPA receptor phosphorylation by the neuropeptide PACAP38

Basal levels of AMPA receptor GluA1 subunit phosphorylation at threonine 840 and serine 845 in hippocampal neurons

Dynamic Regulation of AMPAR Phosphorylation In Vivo Following Acute Behavioral Stress

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