Differential Regulation of Kainate Receptor Trafficking by Phosphorylation of Distinct Sites on GluR6

Katherine W. Roche,Howard Jaffe,Yukiko Nasu-Nishimura,John T.R. Isaac

doi:10.1074/jbc.m109.081141

Abstract

Kainate receptors are widely expressed in the brain, and are present at pre- and postsynaptic sites where they play a prominent role in synaptic plasticity and the regulation of network activity. Within individual neurons, kainate receptors of different subunit compositions are targeted to various locations where they serve distinct functional roles. Despite this complex targeting, relatively little is known about the molecular mechanisms regulating kainate receptor subunit trafficking. Here we investigate the role of phosphorylation in the trafficking of the GluR6 kainate receptor subunit. We identify two specific residues on the GluR6 C terminus, Ser(846) and Ser(868), which are phosphorylated by protein kinase C (PKC) and dramatically regulate GluR6 surface expression. By using GluR6 containing phosphomimetic and nonphosphorylatable mutations for these sites expressed in heterologous cells or in neurons lacking endogenous GluR6, we show that phosphorylation of Ser(846) or Ser(868) regulates receptor trafficking through the biosynthetic pathway. Additionally, Ser(846) phosphorylation dynamically regulates endocytosis of GluR6 at the plasma membrane. Our findings thus demonstrate that phosphorylation of PKC sites on GluR6 regulates surface expression of GluR6 at distinct intracellular trafficking pathways, providing potential molecular mechanisms for the PKC-dependent regulation of synaptic kainate receptor function observed during various forms of synaptic plasticity.

Highlights

Kainate receptors are ionotropic glutamate receptors, which are expressed at both pre- and post-synaptic sites throughout the central nervous system
GluR6-containing kainate receptors are expressed throughout the brain and their function and surface expression dynamically regulated by synaptic activity
We show that protein kinase C (PKC) phosphorylates the GluR6 C terminus on multiple residues including Ser846 and Ser868

Summary

EXPERIMENTAL PROCEDURES

Cells—HeLa cells (ATCC) were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 1% L-glutamine. Transfected live cells were washed once in PBS and labeled with anti-FLAG antibody for 30 min on ice for HeLa cells and at room temperature for neurons. After being washed with PBS, the cells were labeled with fluorescence-conjugated secondary antibody (Alexa 488-conjugated secondary antibody for surface expression analysis in HeLa cells or Alexa 568-conjugated secondary antibody for surface expression analysis in neurons and internalization assays; Molecular Probes) for 45 min at room temperature for visualizing surface-expressed GluR6 and with anti-mouse IgG (1:50; Sigma) for 1 h at room temperature for staining the remaining unlabeled GluR6 on the cell surface. For the surface expression assay, the cells were again labeled with anti-FLAG antibody for 45 min at room temperature. After being washed with PBS, cells were incubated with phycoerythrin-conjugated antimouse IgG1 secondary antibody for 30 min at room temperature and the total expression level of GluR6 was analyzed by FACS as described above. The immunoprecipitates were in vitro phosphorylated as described above and visualized by PhosphorImager analysis

RESULTS

DISCUSSION

We now show that phosphorylation of specific residues on the