Abstract
In the mammalian brain, acetylcholinesterase (AChE) is anchored in cell membranes by a transmembrane protein PRiMA (proline-rich membrane anchor). We present evidence that at least part of the PRiMA-linked AChE is integrated in membrane microdomains called rafts. A significant proportion of PRiMA-linked AChE tetramers from rat brain was recovered in raft fractions; this proportion was markedly higher at low rather than at high concentrations of cold Triton X-100. The detergent-resistant fraction increased during brain development. In NG108-15 neuroblastoma cells transfected with cDNAs encoding AChE(T) and PRiMA, PRiMA-linked G(4) AChE was found in membrane rafts and showed the same sensitivity to cold Triton X-100 extraction as in the brain. The association of PRiMA-linked AChE with rafts was weaker than that of glycosylphosphatidylinositol-anchored G(2) AChE or G(4) Q(N)-H(C)-linked AChE. It was found to depend on the presence of a cholesterol-binding motif, called CRAC (cholesterol recognition/interaction amino acid consensus), located at the junction of transmembrane and cytoplasmic domains of both PRiMA I and II isoforms. The cytoplasmic domain of PRiMA, which differs between PRiMA I and PRiMA II, appeared to play some role in stabilizing the raft localization of G(4) AChE, because the Triton X-100-resistant fraction was smaller with the shorter PRiMA II isoform than that with the longer PRiMA I isoform.
Highlights
Acetylcholinesterase (AChE;2 EC 3.1.1.7) controls synaptic and neurohumoral cholinergic activity by hydrolyzing acetylcholine
In NG108-15 neuroblastoma cells transfected with cDNAs encoding AChET and PRiMA, PRiMA-linked G4 AChE was found in membrane rafts and showed the same sensitivity to cold Triton X-100 extraction as in the brain
The cytoplasmic domain of PRiMA, which differs between PRiMA I and PRiMA II, appeared to play some role in stabilizing the raft localization of G4 AChE, because the Triton X-100-resistant fraction was smaller with the shorter PRiMA II isoform than that with the longer PRiMA I isoform
Summary
The AChER variant produces a soluble monomer that is reported to be up-regulated in the brain during stress [3, 4]; the AChEH variant produces a glycosylphosphatidylinositol (GPI)-anchored dimer that is mainly expressed in blood cells, and the AChET variant, characterized by a 40-residue C-terminal “t” peptide, forms a number of oligomeric assemblies [5]. AChET subunits are predominant in all cholinergic tissues and represent the physiologically active variant; these subunits form tetramers associated with the collagen ColQ and with the transmembrane protein PRiMA (proline-rich membrane anchor), which allow their functional localization. The cytoplasmic domain of PRiMA II has only 11 residues and does not contain these serines Both PRiMA I and PRiMA II can anchor AChET tetramers in cell membranes [13, 14]. The proportion of high detergent-resistant AChE in rafts increased during brain development, suggesting that it may fulfill a possible physiological function. The longer intracellular domain of PRiMA I seems to participate in the stability of this association, because the proportion of raft-associated AChE at high detergent concentration was higher with PRiMA I than with PRiMA II
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