Article Figures & Data
Figures
- Figure 1.
Neuronal activity specifically promotes GluA2 ubiquitination. A, Cortical neurons were incubated in ACSF before bicuculline was added for 15 min. Cells were lysed in RIPA or 1% SDS as described under Materials and Methods. AMPARs were immunoprecipitated using nonimmune IgG-, GluA1-, or GluA2-specific antibodies, and immunoblotted for ubiquitin, GluA1, GluA2, and actin. B, Cortical neurons were incubated in ACSF before bicuculline was added for the indicated time. Cells were lysed, and GluA2 was immunoprecipitated using an anti-GluA2 antibody, and immunoblotted for ubiquitin. C, Cortical neurons were incubated in the absence (−) or presence of bicuculline for 30 min at 37°C in ACSF. Bicuculline was washed and neurons in fresh ACSF were returned to 37°C for recovery periods of 15 or 30 min. Cells were lysed and GluA2 was immunoprecipitated using an anti-GluA2 antibody. Samples were subjected to SDS-PAGE followed by immunoblotted with the indicated antibodies.
- Figure 2.
Activity-dependent GluA2 ubiquitination requires AMPAR activation and calcium. A, Cortical neurons were incubated at 37°C in ACSF for 30 min with the indicated inhibitors before bicuculline (+) was added for 20 min at 37°C. Cells were lysed and GluA2 was immunoprecipitated using an anti-GluA2 antibody. B, Cortical neurons were incubated in the absence (CTL) or presence of glutamate receptor agonists (100 μm AMPA, 30 μm NMDA, 10 μm kainate, or 100 μm DHPG) for 10 min in ACSF. Cells were lysed, and GluA2 was immunoprecipitated with an anti-GluA2 antibody. The asterisk (*) indicates a nonspecific band. C, Cortical neurons were incubated at 37°C in ACSF for 30 min with the indicated inhibitors before adding (+) or omitting (−) AMPA for 10 min at 37°C. Cells were lysed, and GluA2 was immunoprecipitated using an anti-GluA2 antibody. D, Cortical neurons were incubated in ACSF containing calcium or EGTA in the absence (−) or presence (+) of AMPA for 10 min at 37°C. Neurons were lysed, and GluA2 was immunoprecipitated. E, Cortical neurons were incubated at 37°C in ACSF for 60 min in the absence (−) or presence (+) of BAPTA/AM. Cells were washed and returned to 37°C for 15 min in fresh ACSF. Neurons were treated without (−) or with (+) AMPA for 10 min. Cells were lysed, and GluA2 was immunoprecipitated.
- Figure 3.
Endocytosis of GluA2 is linked to GluA2 ubiquitination. A, Cortical neurons were incubated with endocytosis inhibitors or control (CTL) at 37°C in ACSF. Neurons were incubated in the absence (−) or presence (+) of bicuculline for 20 min at 37°C. Cells were lysed and GluA2 was immunoprecipitated. B, Glutamate released in the synaptic cleft binds NMDARs causing calcium influx and also binds AMPARs, which diffuse out of the PSD, where they are internalized via a clathrin- and dynamin-dependent mechanism. Following fission of the vesicle from the plasma membrane, GluA2 is ubiquitinated in an activity-dependent manner and either trafficked to lysosomes for degradation or deubiquitinated and recycled back to the plasma membrane. The drawing is not scaled.
