| Budget Amount *help |
¥4,110,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
PSD-95 is a member of the membrane-associated guanylate kinase (MAGUK) family, the members of which consist of multiple protein-protein interaction motifs, including one or more copies of a PDZ domain, an src homology 3 (SH3) domain, a bridging region known as a HOOK region, and a guanylate kinase (GK)-like domain. Our previous report suggested the interaction between the postsynaptic scaffolding protein, PSD-95, and calmodulin (CaM). Because CaM is reportedly involved in the heteromeric assembly of SAP102 and PSD-95, the association of CaM with PSD-95 within neurons may play a role in switching the MAGUK proteins from intramolecular interactions to intermolecular interactions, which could induce a reorganization of postsynaptic density proteins. Our purpose is to evaluate the intracellular interaction between MAGUKs and Ca^<2+>/CaM, and the switching from the intermolecular interaction to the intramolecular interaction of MAGUKs. We tried to establish the FRET (Fluorescence Resonant Energy Transfer) experiment to monitor the molecular interaction. We have examined the construct, CFP-CaM-M13-YFP, which is known as Ca^<2+> sensor to determine the conditions of the confocal laser scanning microscope (CLSM) for FRET. We succeeded to detect FRET of CFP-CaM-M13-YFP in CaCl_2-containing buffer by fluorescence spectrophotometer (the ratio of acceptor/donor in presence and absence of Ca^<2+> was 60%), and also detect FRET of CFP-CaM-M13-YFP in the cells, when intracellular Ca^<2+> was elevated, by CLSM. Then we made mVenus-MAGUK (binding domain)-CaM-CFP cDNA for single molecule FRET, and found weak FRET signal of this protein in CaClz-containing buffer by fluorescence spectrophotometer (the ratio of acceptor/donor in presence and absence of Ca^<2+> was 20-30%).
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