Acetylcholinesterase (AChE) exists as tetrameric globular (G₄) form enzyme on the membrane of muscle and neuron. This G₄ AChE possesses important physiological functions in cholinergic neurotransmission. PRiMA (P̲roline-R̲i̲ch M̲embrane A̲nchor) cDNA encoding the AChE membrane anchor has been identified and cloned. PRiMA is a transmembrane protein containing a PRAD (P̲roline-R̲ich A̲ttachment D̲omain) domain for interacting with four AChE catalytic subunits (AChE_T), and anchor the enzyme at the surface of muscle and neuron. However, the expression and localization of PRiMA in both muscle and neuron have not been investigated yet. ₄...[ Read more ]

Acetylcholinesterase (AChE) exists as tetrameric globular (G₄) form enzyme on the membrane of muscle and neuron. This G₄ AChE possesses important physiological functions in cholinergic neurotransmission. PRiMA (P̲roline-R̲i̲ch M̲embrane A̲nchor) cDNA encoding the AChE membrane anchor has been identified and cloned. PRiMA is a transmembrane protein containing a PRAD (P̲roline-R̲ich A̲ttachment D̲omain) domain for interacting with four AChE catalytic subunits (AChE_T), and anchor the enzyme at the surface of muscle and neuron. However, the expression and localization of PRiMA in both muscle and neuron have not been investigated yet.

In the current study, mouse C2C12 muscle cells and cultured rat cortical neurons were employed as models to investigate PRiMA regulation. The G₄ AChE was expressed predominantly in the C2C12 myoblasts and mature neurons. The expression of PRiMA mRNA was decreased during the myogenesis of C2C12 muscle cells, and increased during differentiation of cultured neurons. Moreover, the assembly of G₄ AChE could be driven by over expressing PRiMA and AChE_T cDNAs in both cell types. Therefore, the elucidation of regulatory mechanisms of PRiMA expression would be important to understand the precise control of G₄ AChE. Because of the lack of a promising anti-PRiMA antibody, transcriptional regulation of PRiMA was investigated in this study. Results revealed that PRiMA mRNA in muscle was suppressed by MRFs (myogenic regulatory factors); as well as nerve-derived factors, CGRP (calcitonin gene-related peptide) and muscular activity, via the activation of CREB (cAMP-responsive element binding protein). During the differentiation of cultured cortical neurons, the activity of MAPK (mitogen-activated protein kinase) and CaMKII (calcium/calmodulin-dependent protein kinase II) could be induced. The expression of PRiMA mRNA was increased by activating these two signaling pathways; these results suggested the possible roles of MAPK and CaMKll in up regulating PRiMA expression in neurons. Similar results were observed when using the PRiMA promoter in promoter-luciferase-reporter systems of both muscle and neuron cells. These results, therefore, indicated the control of PRiMA mRNA expression was, at least, at a transcriptional level.

Besides a sufficient expression level, a proper positioning of AChE is vital for its synaptic function. The subcellular localization of AChE in brain indicated that G₄ AChE existed in lipid raft micro-domain. In mature cortical neurons, AChE was co-localized with synaptic proteins. The restricted localization of AChE in cultured neurons could be possibly directed by PRiMA. To further reveal the role of PRiMA in directing G₄ AChE to lipid raft, a recombinant system of NG108-15 neuroblastoma cells was employed. In AChE_T and PRiMA co-expressed NG108-15 cells, G₄ AChE was predominantly assembled and localized at lipid raft; the co-expression subsequently caused a dramatic increase of the enzymatic activity. These emerging lines of evidence suggest that PRiMA is responsible not only for enhancing AChE activity, but also for its synaptic targeting on the plasma membrane. Based on these findings, we propose that PRiMA plays vital roles in controlling the expression, enzymatic activity and proper positioning of G₄ AChE in muscles and neurons.