The timely and appropriate trafficking of neurotransmitter receptors is fundamentally important to brain function, including synaptic plasticity. AMPA receptors mediate most of the fast excitatory neurotransmission in the central nervous system and are highly mobile proteins that undergo constitutive and activity-dependent insertion into and removal from synapses, which is thought to be the principal mechanism of synaptic plasticity. Meanwhile, the trafficking of inhibitory GABAB receptors is also involved in regulating synaptic plasticity, but the mechanisms underlying this regulation remain to be revealed. Receptor trafficking is heavily regulated by neuronal activation induced by neurotransmitters or other stimulations. We find that in primary cultured cortical neuron, elevated neuronal activity by activating NMDA receptors in different ways (chem-LTD, chem-LTP, Oxygen/Glucose deprivation) caused changed surface expression of GABABRs or AMPARs by cell surface biotinylation, indicating that both GABABRs and AMPARs undergo activation-dependent trafficking. In addition, activating AMPARs with agonist led to lysosomal-dependent internalization and degradation, providing a potential mechanism of loss of AMPARs during synaptic plasticity. Ubiquitination is a posttranslational modification, which involves conjugation of a 76-amino-acid protein ubiquitin to a lysine residue of the substrate protein. Ubiquitination mediates distinct cellular functions, including degradation. In the present study, ubiquitination of both GluA 1 and GluA2 was evaluated by immunoprecipitation. Interestingly, activation of NMDAR or AMPAR induced GluA2 ubiquitination in primary cultured cortical neuron; at the same time, ubiquitination of overexpressed GluA2 in HEK293 cell model can be accumulated through inhibiting protein degradation. These results suggest activation-dependent GluA2 ubiquitination is a key event in ligand binding-caused internalization and degradation. Ubiquitin-tagged protein cargoes in endosome membrane are sorted to lysosome for degradation by ESCRT (endosomal sorting complex required for transport) machinery. Here we also show that in HEK293 cell model, inhibition of TSG1 01, a member of ESCRT, by siRNA leads to an increased expression of AMPARs and accumulation of ubiquitinated GluA2. This observation infers that ESCRT plays a role in ubiquitination-regulated AMPAR degradation. The present study confirms the importance of ubiquitination to AMPAR trafficking. Ubiquitination-regulated receptordegradation through ESCRT machinery provides a mechanism for activation-dependent receptor trafficking.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:628990 |
| Date | January 2013 |
| Creators | Luo, Jia |
| Publisher | University of Bristol |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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