PSD-95, DLG, ZO-1 (PDZ) domain-mediated protein interactions have been shown to play important roles in the regulation of glutamate receptor function at excitatory synapses. Recent studies demonstrating the rapid regulation of AMPA receptor function during synaptic plasticity have suggested that AMPA receptor interaction with PDZ domain-containing proteins may be dynamically modulated. Here we show that PKC phosphorylation of the AMPA receptor GluR2 subunit differentially modulates its interaction with the PDZ domain-containing proteins GRIP1 and PICK1. The serine residue [serine-880 (Ser880)] in the GluR2 C-terminal sequence (IESVKI) critical for PDZ domain binding is a substrate of PKC and is phosphorylated in vivo. In vitro binding and coimmunoprecipitation studies show that phosphorylation of serine-880 within the GluR2 PDZ ligand significantly decreases GluR2 binding to GRIP1 but not to PICK1. Immunostaining of cultured hippocampal neurons demonstrates that the Ser880-phosphorylated GluR2 subunits are enriched and colocalized with PICK1 in the dendrites, with very little staining observed at excitatory synapses. Interestingly, PKC activation in neurons increases the Ser880 phosphorylation of GluR2 subunits and recruits PICK1 to excitatory synapses. Moreover, PKC stimulation in neurons results in rapid internalization of surface GluR2 subunits. These results suggest that GluR2 phosphorylation of serine-880 may be important in the regulation of the AMPA receptor internalization during synaptic plasticity.

The interaction of PDZ domain-containing proteins with the C termini of ␣-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) receptors has been suggested to be important in the regulation of receptor targeting to excitatory synapses. Recent studies have shown that the rapid internalization of AMPA receptors at synapses may mediate, at least in part, the expression of long-term depression (LTD). We have previously shown that phosphorylation of Ser-880 on the AMPA receptor GluR2 subunit differentially regulated the interaction of GluR2 with the PDZ domain-containing proteins GRIP1 and PICK1. Here, we show that induction of LTD in hippocampal slices increases phosphorylation of Ser-880 within the GluR2 C-terminal PDZ ligand, suggesting that the modulation of GluR2 interaction with GRIP1 and PICK1 may regulate AMPA receptor internalization during LTD. Moreover, postsynaptic intracellular perfusion of GluR2 C-terminal peptides that disrupt GluR2 interaction with PICK1 inhibit the expression of hippocampal LTD. These results suggest that the interaction of GluR2 with PICK1 may play a regulatory role in the expression of LTD in the hippocampus. G lutamate receptors are the major excitatory neurotransmitter receptors in the central nervous system and play critical roles in synaptic plasticity, neuronal development, and neuropsychiatric disorders (1-6). Recent studies have suggested that synaptic targeting and clustering of glutamate receptors are regulated by their interaction with neuronal proteins. Specifically, the interaction of the C termini of specific N-methyl-Daspartate (NMDA) and ␣-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) receptor subunits with PDZ domaincontaining proteins seems to be important for the synaptic localization of the receptors (7-9). AMPA receptor GluR2͞3 subunits have been shown to interact with PDZ domains within the GRIP1, GRIP2͞ABP, and PICK1 proteins through their C-terminal four amino acids (ϪSVKI) (10-15). This interaction has been implicated in the regulation of the synaptic targeting of AMPA receptors because PICK1 coexpression with GluR2 induces clustering of GluR2 (10,13,14) and overexpression of the GluR2 C-terminal PDZ ligand disrupts AMPA receptor synaptic targeting (10).Recent studies have indicated that rapid changes in synaptic efficacy such as those that occur during long-term potentiation (LTP) and long-term depression (LTD) may be caused by rapid changes in AMPA receptor function. Although some of these effects may be mediated by modulation of AMPA receptor function by phosphorylation (16-19), rapid changes in the levels of synaptic AMPA receptors may also mediate changes in synaptic efficacy (20)(21)(22)(23)(24)(25)(26)(27). One possible mechanism for the rapid modification of the synaptic targeting of AMPA receptors is through the dynamic regulation of AMPA receptor-PDZ domain interactions. We have previously shown that phosphorylation of Ser-880 within the GluR2 C-terminal PDZ ligand differentially regulates its interaction with the PDZ domains of GRIP1 and PICK1 ...

Cerebellar LTD requires activation of PKC and is expressed, at least in part, as postsynaptic AMPA receptor internalization. Recently, it was shown that AMPA receptor internalization requires clathrin-mediated endocytosis and depends upon the carboxy-terminal region of GluR2/3. Phosphorylation of Ser-880 in this region by PKC differentially regulates the binding of the PDZ domain-containing proteins GRIP/ABP and PICK1. Peptides, corresponding to the phosphorylated and dephosphorylated GluR2 carboxy-terminal PDZ binding motif, were perfused in cerebellar Purkinje cells grown in culture. Both the dephospho form (which blocks binding of GRIP/ABP and PICK1) and the phospho form (which selectively blocks PICK1) attenuated LTD induction by glutamate/depolarization pairing, as did antibodies directed against the PDZ domain of PICK1. These findings indicate that expression of cerebellar LTD requires PKC-regulated interactions between the carboxy-terminal of GluR2/3 and PDZ domain-containing proteins.