Tetrameric globular acetylcholinesterase (G
4 AChE), assembled by the AChE catalytic subunit and PRiMA (P̲roline-R̲ich M̲embrane A̲nchor), is located on the plasma membrane of muscle and neuron for cholinergic transmission. In this study, the analysis of PRiMA in terms of expression, membrane localization and interaction would be revealed.
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Tetrameric globular acetylcholinesterase (G
4 AChE), assembled by the AChE catalytic subunit and PRiMA (P̲roline-R̲ich M̲embrane A̲nchor), is located on the plasma membrane of muscle and neuron for cholinergic transmission. In this study, the analysis of PRiMA in terms of expression, membrane localization and interaction would be revealed.
During the development of rat muscles, PRiMA and AChE mRNAs, and the enzymatic activity increased dramatically. G
4 AChE in muscle was recognized specifically by a PRiMA antibody, indicating the association of this enzyme with PRiMA. Using Western blot and ELISA, both PRiMA protein and PRiMA-linked G
4 AChE level in fast-twitch muscle were higher than that in slow-twitch muscle. In parallel, the expression of PRiMA, AChE and G
4 AChE also increased in the spinal cord during development. After denervation, the expression of PRiMA, AChE and G
4 AChE decreased markedly in the spinal cord, and in fast- and slow-twitch muscles, indicating that both muscles and motor neuron contributed to the synaptic expression of G
4 AChE.
Regarding to membrane localization, G
4 AChE was present in a microdomain called membrane raft where signalling molecules were clustered. The association of G
4 AChE with membrane raft in rat cerebrum was sensitive to detergent concentration. The cholesterol-binding motif (CRAC) of PRiMA was shown to participate in cholesterol interaction in membrane raft. Treatment of cholesterol-depletion agent and CRAC mutation on PRiMA were able to eliminate the raft localization, indicating that the CRAC motif was responsible for membrane raft localization of PRiMA. Finally, the interaction of G
4 AChE and V-ATPase in membrane raft was revealed by the proteomic approach. Such interaction might be involved via the cytoplasmic domain of PRiMA.
In summary, we concluded that the PRiMA-linked G
4 AChE was present at the synaptic region, and the membrane raft localization of this enzyme was mediated by the CRAC domain.
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