Functional Investigation Of PICK1- Interacting Proteins In ASICs And AMPA Receptor Trafficking

Background:PICK1 (Protein that interacts with C kinase 1) is a peripheral membrane protein that identified as one of the protein kinase C-a (PKCa) interacting protein. PICK1 can bind to a lot of membrane proteins. For example, PICK1 can bind to not only the ASIC (acid-sensing ion channels) but also GluA2 which is one of the AMPA (a-amino-3-hydroxy-5-methylisoxazoloe-4-propionic acid-type) receptor subunit, and mediate their cellular translocations and surface expression. PICK1 bi-directionally facilitates AMPA receptor’s removing from and incorporation onto synaptic membranes during long-term depression and long-term potentiation. Under basal activity, PICK1 not only mediates AMPA receptor trafficking in synaptic targeting and membrane expression, but also regulates ASIC trafficking and surface expression level. According to a lot of evidence, PICK1 binds to ASIC la and ASIC2a directly, then regulates cytotoxicity by affecting their surface expression level, and finally regulates cell apoptosis. However, it is not clear that how PICK1 regulates the cytotoxicity. Moreover, PICK1 forms heteromeric complex with ICA69 (Islet Cell Autoantigen 69 kDa) and negatively regulates the synaptic translocation of AMPA receptor. However, the underlying mechanism on how PICK1-ICA69 complex regulates AMPA receptor trafficking is still unclear. Our results show that KIF2C binds to PICK1 directly, and forms a complex with ICA69 and AMPA receptor. We further show that KIF2C can regulate the membrane expression of AMPA receptor.Objective:To identify novel PICK1 binding partners and investigate their functions. On one hand, test whether the syntabulin-PICK1 interaction regulates neuronal apoptosis. On the other hand, investigate whether the KIF2C-PICK1 interaction affects the AMPA receptor distribution. To explain the mechanism of ASIC and AMPA receptor trafficking, finally to examine the mechnism of how ASIC channels regulate neuronal apoptosis and how AMPA receptor trafficking.Methods:We found the syntabulin as a novel protein interacting with PICK1 by yeast-two hybrid. And we found another candidate protein KIF2C interacting with PICK1 and ICA69 by Co-Immunoprecipitation and mass spectrometry. Through western-blot and immunocytochemistry approaches, we further confirmed their interactions. Finally, we tested the function of syntabulin on surface expression of ASIC and neuronal apoptosis. We also tested the function of KIF2C on the localization and surface expression of AMPA receptor in heterologous cell system and neurons.Results:(1) According to our results, under acidic condition, knocking down syntabulin increased neuronal apoptosis in hipocampal neurons. By biotinylation, we also found that surface expression level of ASIC2 decreased and total expression level of ASIC2 increased after knocking down syntabulin in hippcampal neurons. The in vitro co-immunoprecipitation assay showed that syntabulin could form a complex with ASIC1a and ASIC2a through PICK1. However, syntabulin preferred bind to more ASIC2a than ASICla. Moreover, syntabulin co-localized with ASIC2a through PICK1. But no matter there was PICK1 or not, syntabulin didn’t co-localize with ASIC1a. (2) The mass spectrometry result identified 19 positive proteins interacting with PICK1, and 15 with ICA69. Among them, KIF2C was proved to bind PICK1 directly, and form a complex with ICA69 through PICK1. The interaction between PICK1 and KIF2C didn’t affect the binding affinity and co-clusters formation between PICKl and GluA2. Under physiological conditions, KIF2C dominately distributed in the brain and testes, and interacted with GluA2 in brain. We further proved that KIF2C formed complex with GluA2 through PICKl. In 293T cells, the surface GluA2 level was reduced by overexpressing KIF2C. When KIF2C and PICK1 were co-epexpressed, the surface level of GluA2 showed a further reduction. This could be a result by the synergetic microtubule depolymerization effect of KIF2C’s and GluA2 surface removing effect of PICKl. In cultured hippocampal neurons, KIF2C could co-localize with PSD-95 and GluA2, and reduce GluA2 surface expression level.Conclusion:(1) Our results show that syntabulin can form a complex with ASIC channels through PICK1, then regulates the surface and total expression level of ASIC2, and finally mediates the neuronal acidtoxicity. (2) Our results show that KIF2C binds PICK1 directly, and forms a complex with GluA2. KIF2C can regulate the AMPA receptor trafficking depending on the interaction with PICKl and depolymerizing of microtubules.