C-terminal Domain Of ICA69Interacts With PICK1and Actson Trafficking Of PICK1-PKCα Complex And Cerebellar Plasticity

Bachground: PICK1(protein interacting with C-kinase1) is a PKC (protein kinase C)-binding protein, which is essential for synaptic plasticity. The trafficking of PKCa-PICK1complex to plasma membrane is critical for the internalization of GluR2and induction of long-term depression(LTD). ICA69(islet cell autoantigen69kDa) is identified as a major binding partner of PICK1. In physiological conditions in tissue in vivo, the majority of PICK1and ICA69form heterologous complexes. Besides BAR domain, ICA69also contains a C-terminal domain (ICAC, ICA69amino acids257-480), which shows no apparent homology to other known proteins. Transfection of ICA69in cultured neurons redistributes PICK1from synapses to dendrites. While heteromeric BAR domain complex is suggested to underlie the interaction between PICK1and ICA69, the role of ICAC in PICK1-ICA69complex is unknown. Our experiment result show that the ICAC domain can bind PICK1strongly, and the domain maybe also have a significant impact on PICK1function. Objective:to detect whether ICA69or ICAC domain affect the membrane transport and the cerebellar plasticity, furthermore to search the underlied mechanisms. To look for the factors determine binding capacity between ICA69and PICK1, in order to make out how PICK1be regulated by ICA69in different condition. To provide ideas for designing peptide drug targeting to PICK1.Methods: to gain insights of PICK1-ICA69complex, we tested the interaction between ICAC or its truncations and PICK1using Co-IP, Western Blot, immunocytochemistry, and fluorescence resonance energy transfer (FRET) assays.In living cells, by confocal microscope to real-time observe TPA-activated fluorescent protein movement, by electrophysiological techniques to detect whether the different domains of ICA69affects the LTD cerebellar Purkinje cells or not.Results: We found that ICAC was also able to strongly interact with PICK1besides BAR domain and interaction of PICK1-ICA69was related to phosphorylation, dephosphorylation and [Ca2+]. The trafficking of the fluorescent proteins were observed after transfection in living cell using time-lapse confocal microscopy. We found that either ICA69or ICAC interacted with PICK1and regulated the trafficking of PICK1-PKCa complex. ICAC and AICAC (containing BAR domain) might function distinctly in the association of ICA69with PICK1. While AICAC domain inclined to form clusters, the distribution of ICAC was diffuse. The trafficking of PICK1to plasma membrane mediated by activated PKCa that was inhibited by ICA69. This action might ascribe to ICAC, because overexpression of ICAC, but not AICAC, interrupted PKCa-mediated PICKl trafficking. Notably, infusion of maltose binding protein (MBP) fusion protein, MBP-ICA69or MBP-ICAC, but not MBP or MBP-AICAC in cerebellar Purkinje cells significantly inhibited the induction of LTD at parallel fiber-and climbing fiber-Purkinje cell synapses.Conclusion: Our experiments showed that ICAC is an important domain for the ICA69-PICK1interaction and ICAC inhibited the trafficking of PICK1to the plasma membrane stimulated by activated PKCa and cerebellar LTD.