Neto1 and Neto2 are CUB domain-containing transmembrane proteins that are expressed in the mammalian brain. Previous studies showed that Neto1 is a NMDAR-associated protein with important roles in synaptic plasticity and learning/memory (Ng et al., 2009). To establish the functions of Neto2, I first searched for its binding partners. Using yeast two-hybrid analysis, GST pull-down and co-immunoprecipitation studies, I found that Neto2 can bind to the PDZ domain-containing protein GRIP. In the brain, GRIP regulates the synaptic trafficking and stability of AMPA and kainate receptors (KARs) (Hirbec et al., 2003). To determine whether Neto2 is required for the synaptic expression of KARs and/or AMPARs, I examined whether Neto2 was associated with these receptors at the postsynaptic membrane. Coimmunoprecipitation studies showed that while Neto2 is a component of postsynaptic KAR protein complexes, it is not associated with AMPARs. In the cerebellum, Neto2-null mice showed a 44% (n=3;p<0.01) decrease in the abundance of postsynaptic KARs with no change in the level of total KARs, thus suggesting a specific deficit in KAR synaptic localization. Unexpectedly, loss of Neto2 had no effect on the abundance of hippocampal KARs (n=3; p>0.05), or on neurotransmission by them (n=12; p>0.05). To determine whether this normal KAR function might be due to compensation by Neto1, which also interacts with KARs, I examined KAR abundance in Neto1-null, and Neto1/2-double null hippocampus. Loss of Neto1 resulted in a 53% decrease in postsynaptic levels of GluK2-KARs (n=3;p<0.01). However, in double null animals, the reduction was indistinguishable from Neto1 single null mice, suggesting that Neto2 is not involved in the postsynaptic localization of hippocampal KARs. In Neto1-null mice, KAR-mediated currents showed smaller amplitude (61% of wild-type;n=14;p<0.01), and faster decay kinetics (40% of wild-type;n=14;p<0.001). Together, these findings establish both Neto1 and Neto2 as auxiliary proteins of native KARs: Neto1 regulates the synaptic abundance and kinetics of KARs in the hippocampus, while Neto2 mediates the synaptic localization of cerebellar KARs. Additionally, the results presented here, in conjunction with previous findings, reveal a unique ability of Neto1 in controlling synaptic transmission by serving as an auxiliary protein for two different classes of ionotropic glutamate receptors.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/36014 |
| Date | 13 August 2013 |
| Creators | Tang, Man |
| Contributors | McInnes, Roderick |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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