Phosphorylation of Glutamate Receptor Interacting Protein 1 Regulates Surface Expression of Glutamate Receptors

Karina Kulangara,Michel Kropf,Liliane Glauser,Sarah Magnin,Stefano Alberi,Alexandre Yersin,Harald Hirling

doi:10.1074/jbc.m606471200

Abstract

The number of synaptic alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors (AMPARs) controls the strength of excitatory transmission. AMPARs cycle between internal endosomal compartments and the plasma membrane. Interactions between the AMPAR subunit GluR2, glutamate receptor interacting protein 1 (GRIP1), and the endosomal protein NEEP21 are essential for correct GluR2 recycling. Here we show that an about 85-kDa protein kinase phosphorylates GRIP1 on serine 917. This kinase is present in NEEP21 immunocomplexes and is activated in okadaic acid-treated neurons. Pulldown assays and atomic force microscopy indicate that phosphorylated GRIP shows reduced binding to NEEP21. AMPA or N-methyl-D-aspartate stimulation of hippocampal neurons induces delayed phosphorylation of the same serine 917. A wild type carboxy-terminal GRIP1 fragment expressed in hippocampal neurons interferes with GluR2 surface expression. On the contrary, a S917D mutant fragment does not interfere with GluR2 surface expression. Likewise, coexpression of GluR2 together with full-length wild type GRIP1 enhances GluR2 surface expression in fibroblasts, whereas full-length GRIP1-S917D had no effect. This indicates that this serine residue is implicated in AMPAR cycling. Our results identify an important regulatory mechanism in the trafficking of AMPAR subunits between internal compartments and the plasma membrane.

Highlights

Eral diffusion to and from synapses are key mechanisms that govern synaptic strength [1,2,3,4,5]
The interaction between GluR2 and type II PDZ domain proteins like protein interacting with C kinase 1 (PICK1), glutamate receptor interacting protein (GRIP), and AMPA receptor (AMPAR)-binding protein (ABP) is necessary for this cycling [17]
These results indicate that both the extract we hypothesized that a kinase that phosphorylates GRIP might from okadaic acid (OA)-treated neurons and the anti-NEEP21 immune pellet be associated with this complex

Summary

EXPERIMENTAL PROCEDURES

Antibodies—Antibodies against the following proteins were applied for Western blotting (W), immunofluorescence (IF), or immunoprecipitation: monoclonal antibodies GRIP (W, 1:10000; IF, 1:300; BD Biosciences), GluR2 (W, 1:100; IF, 1:10, BD Biosciences), actin (W 1:2000, Chemicon, Temecula, CA); polyclonal antibodies GluR1 (IF, 1:5, Calbiochem), GluR2/3 (W 1:1000, Chemicon), NEEP21 (W 1:6000; IF, 1:300 [39]). Immunoprecipitation, Surface Biotinylation, and Cell Fractionation—Preparation of brain membrane extracts from postnatal day 10 rats and covalent cross-linking of antibodies to protein A or G beads were done as previously described [38]. In Vitro Kinase Assay—Fusion proteins were immobilized on glutathione-agarose beads and incubated for 8 min at 30 °C in the presence of [␥-32P]ATP (10 ␮Ci, Amersham Biosciences) in phosphorylation buffer with either 10 ␮g of neuron extract or eluted proteins from IgG or anti-NEEP21-immunoprecipitations. D, total extracts of hippocampal neurons treated as above were analyzed for GluR2 by Western blotting. The total GluR2 content of the neurons was not altered, as verified by Western blotting of OA-treated neurons (Fig. 1D), excluding an effect on gene expression or degradation. GluR2 was localized to synaptic as well as extra-synaptic sites, and this distribution did not change upon OA treatment (data not shown)

To examine the site to which

RESULTS

Findings

DISCUSSION