Studying the protein-protein interactions in the postsynaptic density by means of immunoabsorption and chemical crosslinking |
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Authors: | Chang Chia-Wei Peng Sheng-Chih Cheng Wen-Yao Liu Szu-Heng Cheng Huei-Hsuan Huang San-Yuan Chang Yen-Chung |
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Affiliation: | Institute of Molecular Medicine, Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China. |
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Abstract: | Postsynaptic densities (PSDs), isolated from porcine cerebral cortices, are large disk-shaped aggregates consisting of hundreds of different proteins. To study the protein-protein interactions in such complex supramolecules, we developed a procedure to break up the PSD's overall structure, while preserving some interactions between individual proteins. Using the resulting PSD sample and an indirect immunoabsorption procedure, PSD-95 was isolated along with the α- and β-subunits of calcium calmodulin-dependent protein kinase II (CaMKIIα and CaMKIIβ), α-tubulin, β-tubulin, and Chapsyn110. Similarly, CaMKIIα was isolated along with CaMKIIβ, α-tubulin, β-tubulin, and small amounts of PSD-95. The proteins isolated from PSDs treated with a cleavable bifunctional crosslinking reagent were further subjected to diagonal gel electrophoresis analysis, and the results indicated that CaMKIIα resides next to α-tubulin in the PSD. Overall, the results obtained here suggest that within the PSD, large aggregates of CaMKIIα, CaMKIIβ, α-tubulin, and β-tubulin may occur that indirectly associate with PSD-95 and Chapsyn110. Such a protein organization would allow interactions with F-actin in the cytoplasm and with proteins, such as N-methyl-D-aspartate receptors, which reside on the postsynaptic membrane. Furthermore, it would facilitate binding to proteins such as the various microtubule-associated proteins that reside in the core region of the PSD. |
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