Chung H J, Xia J, Scannevin R H, Zhang X, Huganir R L
Department of Neuroscience, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
J Neurosci. 2000 Oct 1;20(19):7258-67. doi: 10.1523/JNEUROSCI.20-19-07258.2000.
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.
PSD-95、DLG、ZO-1（PDZ）结构域介导的蛋白质相互作用已被证明在兴奋性突触处谷氨酸受体功能的调节中发挥重要作用。最近的研究表明，在突触可塑性过程中AMPA受体功能可被快速调节，这提示AMPA受体与含PDZ结构域蛋白的相互作用可能受到动态调控。在此我们表明，蛋白激酶C（PKC）对AMPA受体GluR2亚基的磷酸化可差异性地调节其与含PDZ结构域蛋白GRIP1和PICK1的相互作用。GluR2 C末端序列（IESVKI）中对PDZ结构域结合至关重要的丝氨酸残基[丝氨酸-880（Ser880）]是PKC的底物，且在体内被磷酸化。体外结合和免疫共沉淀研究表明，GluR2 PDZ配体中丝氨酸-880的磷酸化显著降低GluR2与GRIP1的结合，但不影响其与PICK1的结合。对培养的海马神经元进行免疫染色显示，Ser880磷酸化的GluR2亚基在树突中富集并与PICK1共定位，在兴奋性突触处观察到的染色很少。有趣的是，神经元中PKC的激活增加了GluR2亚基的Ser880磷酸化，并将PICK1募集到兴奋性突触。此外，神经元中PKC刺激导致表面GluR2亚基快速内化。这些结果表明，丝氨酸-880的GluR2磷酸化可能在突触可塑性过程中AMPA受体内化的调节中起重要作用。
