PICK1 and phosphorylation of the glutamate receptor 2 (GluR2) AMPA receptor subunit regulates GluR2 recycling after NMDA receptor-induced internalization.
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.
Lin
Da-Ting
DT
Department of Neuroscience, Johns Hopkins University School of Medicine, Howard Hughes Medical Institute, Baltimore, Maryland 21205, USA.
Huganir
Richard L
RL
D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D052066:N-Ethylmaleimide-Sensitive Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D051381:Rats; D018091:Receptors, AMPA / Q000235:genetics / Q000378:metabolism*; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; C485187:PHluorin; C091822:Prkcabp protein, mouse; D021122:Protein Subunits; D018091:Receptors, AMPA; D016194:Receptors, N-Methyl-D-Aspartate; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins; C498733:Nsf protein, mouse; C104722:glutamate receptor ionotropic, AMPA 2