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

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 lacks 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.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 Correspondence to: Hey-Kyoung Lee. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. , Lee et al., 2003. Recent data suggests that S845 and S818 affect the trafficking AMPA receptors to synapses and/or stabilize synaptic AMPA receptors (Esteban et al., 2003,...

Extensive phosphorylation of AMPA receptors in neurons

“…In our Phos-tag experiments we are able to detect single phosphorylated S845 or S831 species, as well as a more complex population-containing multiple phosphorylations. GluA1 is also known to be phosphorylated at S567 by CaMKII (38), S818 and T840 by PKC (39,40), and more recently at S863 by PAK3 (41). Interestingly, we recently showed that stimulation of neurons with the neuropeptide PACAP38 resulted in coordinated increases in phosphorylation of GluA1 S845 and dephosphorylation of T840 (42), indicating a complex relationship exists between different phosphorylation sites.…”

Specific Roles of AMPA Receptor Subunit GluR1 (GluA1) Phosphorylation Sites in Regulating Synaptic Plasticity in the CA1 Region of Hippocampus

“…Although our data from the S845A mutants are the same as those observed in the GluR1 "double phosphomutants" , it is slightly different from the "penta phosphomutants," which showed LTD deficits only in the adults ). Unlike the "double" or the "single" phosphomutants, the "penta" phosphomutants carry additional three mutations at amino acid residues, including a novel phosphorylation site, T840 (Delgado et al 2007;Lee H-K et al 2007). The lack of T840 apparently rescued the LTD phenotype in juveniles lacking both S831 and S845.…”

Glutamate Receptor Ion Channels: Structure, Regulation, and Function

“…Phosphorylation of the two membrane-proximal PKC sites (SRS 816 ES 818 KR) enhances interaction between the actin-binding 4.1N protein and the GluA1 C-terminal domain, which facilitates insertion of this subunit into the plasma membrane (Boehm et al, 2006;Lin et al, 2009), a mechanism involved in long-term potentiation. Phosphorylation of TST 840 LPR by PKC has been suggested to influence synaptic transmission in an age-dependent fashion (Lee et al, 2007b). Two other GluA1 phosphorylation sites control functional properties of AMPA receptor channels.…”