PICK1 and phosphorylation of the glutamate receptor 2 (GluR2) AMPA receptor subunit regulates GluR2 recycling after NMDA receptor-induced internalization

Abstract

Changes in surface trafficking of AMPA receptors play an important role in synaptic plasticity. Phosphorylation of the C terminus of the AMPA receptor (AMPAR) subunit glutamate receptor 2 (GluR2) and the binding of GluR2 to the PDZ [postsynaptic density-95/Discs large/zona occludens-1]-domain containing protein, protein interacting with protein kinase C (PICK1), have been proposed to play an important role in NMDA receptor dependent internalization of GluR2. However, the fate of internalized GluR2 after NMDA receptor (NMDAR) activation is still unclear. Both recycling and degradation of GluR2 after the activation of NMDAR have been reported. Here, we used a pH-sensitive green fluorescent protein variant, pHluorin, tagged to the N terminus of GluR2 (pH-GluR2) to study the dynamic internalization and recycling of GluR2 after NMDAR activation. Using fluorescence recovery after photobleach (FRAP), we directly demonstrate that internalized pH-GluR2 subunits recycle back to the cell surface after NMDAR activation. We further demonstrate that changing the phosphorylation state of the S880 residue at the C terminus of GluR2 does not affect NMDAR-dependent GluR2 internalization, but alters the recycling of GluR2 after NMDAR activation. In addition, mutation of the N-ethylmaleimide-sensitive fusion protein (NSF) binding site in the pH-GluR2 slows receptor recycling. Finally, neurons lacking PICK1 display normal NMDAR dependent GluR2 internalization compared with wild-type neurons, but demonstrate accelerated GluR2 recycling after NMDAR activation. These results indicate that phosphorylation of GluR2 S880 and NSF and PICK1 binding to GluR2 dynamically regulate GluR2 recycling.

Figure 1.

pH-GluR2 is internalized following NMDAR activation and recycles to the cell surface. A, Representative images of a hippocampal neuron subjected to NMDA perfusion/washout cycle show that NMDA induces loss of significant amount of observed pH-GluR2 fluorescence; washout of NMDA resulted in full return of pH-GluR2 fluorescence over time. B, Time trace of pH-GluR2 fluorescence change in response to 5-min NMDA treatment for the experiment presented in A. C, A hippocampal neuron expressing pH-GluR2 shown (top left). pH-GluR2 on the surface of the cell soma was photobleached immediately before NMDA perfusion, resulting in loss of the majority of pH-GluR2 fluorescence in this somatic area (top right). NMDA perfusion for 5 min induces pH-GluR2 internalization (bottom left). NMDA washout results in full recovery of pH-GluR2 fluorescence in dendritic surface (unbleached area) and recovery of pH-GluR2 fluorescence to postbleached level in somatic surface (bleached area, white square). D, Time trace of fluorescence change in somatic (bleached) surface for the experiment presented in C. E, A hippocampal neuron expressing pH-GluR2 is shown (top left). The pH-GluR2 relocates to endosomal compartments after 5-min NMDA perfusion, resulting in loss of the majority of pH-GluR2 fluorescence (top right). The same photobleach protocol used in C was then applied to the surface of somatic area immediately before NMDA washout, resulting in a small additional loss of pH-GluR2 fluorescence in the somatic area (bottom left). NMDA washout results in complete recovery of pH-GluR2 fluorescence on the dendritic surface (unbleached area) and almost complete recovery of pH-GluR2 fluorescence on the somatic surface (bleached area, white square). F, Time trace of fluorescence change in somatic (bleached) surface for the experiment presented in E. Scale bars: A, C, E, 10 μm.

Figure 2.

Modulating GluR2 S880 phosphorylation state as well as NSF binding to GluR2 alters GluR2 recycling process. A, Selective images of pH-GluR2 wt, S880E, K882A, and ΔNSF during NMDA perfusion/washout experiments. B, Average fluorescence time course for pH-GluR2 wt, S880E, K882A, and ΔNSF mutants during NMDA perfusion/washout experiments. C, Histograms of pHluorin fluorescence change amplitude in response to NMDA and T_1/2 after NMDA washout. Scale bars: 10 μm.

Figure 3.

Neurons derived from PICK1 knock-out mice displays altered GluR2 recycling. A, Selective images of pH-GluR2 from wild-type and PICK1 knock-out hippocampal neurons during NMDA perfusion/washout experiments. B, Average fluorescence time course of pH-GluR2 from wild-type and PICK1 knock-out hippocampal neurons during NMDA perfusion/washout experiments. C, Histograms of pHluorin fluorescence change amplitude in response to NMDA and T_1/2 after NMDA washout. Scale bars: 10 μm.