Identification and characterization of a novel phosphorylation site on the GluR1 subunit of AMPA receptors

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Abstract

Phosphorylation of various AMPA receptor subunits can alter synaptic transmission and plasticity at excitatory glutamatergic synapses in the central nervous system. Here, we identified threonine-840 (T840) on the GluR1 subunit of AMPA receptors as a novel phosphorylation site. T840 is phosphorylated by protein kinase C (PKC) in vitro and is a highly turned-over phosphorylation site in the hippocampus. Interestingly, the high basal phosphorylation of T840 in the hippocampus is maintained by a persistent activity of a protein kinase, which is counter-balanced by a basal protein phosphatase activity. To study the function of T840, we generated a line of mutant mice lacking this phosphorylation site using a gene knock-in technique. The mice generated lack T840, in addition to two previously identified phosphorylation sites S831 and S845. Using this mouse, we demonstrate that T840 may regulate synaptic plasticity in an age-dependent manner.

Introduction

Many of the brain functions critically depend on plasticity of the glutamatergic excitatory synaptic transmission, which can be accomplished postsynaptically by modulation of glutamate receptors. AMPA-type glutamate receptors mediate the majority of fast excitatory synaptic transmission, and functional changes to these receptors are implicated in synaptic plasticity (Song and Huganir, 2002, Collingridge et al., 2004). One way to alter the function of AMPA receptors is by changing phosphorylation of its subunits. All four subunits of AMPA receptors, GluR1–4, which have several identified phosphorylation sites on their intracellular carboxy-terminus (Song and Huganir, 2002).

Among the four subunits, GluR1 has three identified phosphorylation sites, serines 818 (S818) (Boehm et al., 2006), 831 (S831) and 845 (S845) (Roche et al., 1996). S831 is phosphorylated by calcium/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC), S845 is a cAMP-dependent protein kinase (PKA) substrate (Roche et al., 1996, Barria et al., 1997a, Mammen et al., 1997), while S818 is phosphorylated by PKC (Boehm et al., 2006). Changes in phosphorylation of GluR1 at S845 and S831 affect AMPA receptor mediated currents (Derkach et al., 1999, Banke et al., 2000) and are involved in long-term potentiation (LTP) and long-term depression (LTD) in the hippocampus (Barria et al., 1997b, Kameyama et al., 1998, Lee et al., 1998, Lee et al., 2000, Lee et al., 2003). Recent data suggest that S845 and S818 affect the trafficking AMPA receptors to synapses and/or stabilize synaptic AMPA receptors (Esteban et al., 2003, Lee et al., 2003, Boehm et al., 2006).

Phosphorylation of GluR2 subunit at serine-880 has been implicated in LTD both in hippocampus and in cerebellum (Daw et al., 2000, Matsuda et al., 2000, Xia et al., 2000, Kim et al., 2001, Chung et al., 2003). Phosphorylation of this site can regulate synaptic localization of AMPA receptors by altering its interaction with intracellular binding partners: glutamate receptor interacting protein (GRIP)/AMPA receptor binding protein (ABP) and protein interacting with C-kinase-1 (PICK-1) (Matsuda et al., 1999, Chung et al., 2000).

Among several identified GluR4 subunit phosphorylation sites (Carvalho et al., 1999), S842, which is a PKA phosphorylation site, was shown to be involved in activity-dependent trafficking of AMPA receptors to synapses (Esteban et al., 2003). Therefore, phosphorylation of different subunits of AMPA receptor seems to play an important role in regulating receptor function, which can alter synaptic transmission and/or synaptic plasticity.

We had recognized early on from generating phosphopeptide maps of the GluR1 subunit that there remain additional unidentified phosphorylation sites on GluR1 (Blackstone et al., 1994, Roche et al., 1996, Mammen et al., 1997). Here, we report that threonine-840 (T840) is one of the major phosphorylation sites on GluR1. This site is phosphorylated by PKC in vitro and can be regulated by altering PKC activity in hippocampal slices. Interestingly, phosphorylation of T840 undergoes rapid turnover under basal conditions in the hippocampus, sustained by a balance between persistently active protein kinase(s) and protein phosphatase(s). To study the functional role of GluR1 T840, we generated a line of mice lacking T840 and show that it may be involved in regulating synaptic plasticity mechanisms in an age-dependent fashion.

Section snippets

Mapping of a “basal” phosphorylation site on GluR1

To identify phosphorylation sites on GluR1 subunit of AMPA receptors, we generated phosphopeptide maps using trypsin digested GluR1 immunoprecipitated from metabolically labeled hippocampal slices. We were specifically interested in a large phosphopeptide spot (#1 in Fig. 1) that had not been previously characterized. This phosphopeptide was highly phosphorylated under basal conditions and was not noticeably upregulated by either forskolin (50 μM, 30 min) or phorbol ester (TPA: 1 μM, 30 min)

Discussion

Here we identified and described T840 as a novel phosphorylation site on GluR1 subunit of AMPA receptors. GluR1-T840 is phosphorylated by PKC in vitro and is a highly turned-over phosphorylation site under basal conditions in the hippocampus. The basal phosphorylation of GluR1-T840 in vivo is likely due to a persistent activity of a protein kinase, which is counter-balanced by a basal protein phosphatase activity. In order to test the in vivo role of GluR1-T840, we generated a GluR1 “penta”

Hippocampal slice preparation and metabolic labeling

Hippocampal slices from adult Sprague–Dawley rats (150–250 g, males) were prepared as previously described (Lee et al., 2000). In brief, 400 μm thick hippocampal slices were dissected using ice-cold dissection buffer (212.7 mM sucrose, 2.6 mM KCl, 1.23 mM NaH2PO4, 26 mM NaHCO3, 10 mM dextrose, 3 mM MgCl2 and 1 mM CaCl2) and recovered at room temperature in ACSF (124 mM NaCl, 5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 10 mM dextrose, 1.5 mM MgCl2 and 2.5 mM CaCl2) inside a Plexiglas chamber

Acknowledgments

This work was supported by the HHMI and NIH grant to RLH, and the Sloan fellowship and NIH grant to HL.

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    Current address: Molecular Neurobiology Group, Neuroscience Research Institute, National Institute of Advanced Industrial Science and Technology(AIST), Ibaraki 305-8566, Japan.

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