Calnexin-dependent Enhancement of Nicotinic Acetylcholine Receptor Assembly and Surface Expression

Chang, Weise; Gelman, Marina S.; Prives, Joav

doi:10.1074/jbc.272.46.28925

Cited by 73 publications

(65 citation statements)

References 45 publications

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“…We cloned chErp72 because it is a member of the PDI family but does not have a known role in collagen synthesis as does PDI. We also tested canine calnexin because it is an ER molecular chaperone unrelated to the PDI family that is known to enhance acetylcholine receptor maturation by promoting subunit folding and assembly (23)(24)(25)(26). Overexpression of either of these molecular chaperones consistently increased expression of the tetrameric (G4) and collagen-tailed AChE forms (A12-A4) in three independent experiments (Fig.…”

Section: The Levels Of Endoplasmic Reticulum Molecular Chaperones Pdimentioning

confidence: 99%

Limiting Role of Protein Disulfide Isomerase in the Expression of Collagen-tailed Acetylcholinesterase Forms in Muscle

Ruiz¹,

Rotundo

2009

Journal of Biological Chemistry

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The expression of acetylcholinesterase (AChE) in skeletal muscle is regulated by muscle activity; however, the underlying molecular mechanisms are incompletely understood. We show here that the expression of the synaptic collagen-tailed AChE form (ColQ-AChE) in quail muscle cultures can be regulated by muscle activity post-translationally. Inhibition of thiol oxidoreductase activity decreases expression of all active AChE forms. Likewise, primary quail myotubes transfected with protein disulfide isomerase (PDI) short hairpin RNAs showed a significant decrease of both the intracellular pool of all collagentailed AChE forms and cell surface AChE clusters. Conversely, overexpression of PDI, endoplasmic reticulum protein 72, or calnexin in muscle cells enhanced expression of all collagentailed AChE forms. Overexpression of PDI had the most dramatic effect with a 100% increase in the intracellular ColQAChE pool and cell surface enzyme activity. Moreover, the levels of PDI are regulated by muscle activity and correlate with the levels of ColQ-AChE and AChE tetramers. Finally, we demonstrate that PDI interacts directly with AChE intracellularly. These results show that collagen-tailed AChE form levels induced by muscle activity can be regulated by molecular chaperones and suggest that newly synthesized exportable proteins may compete for chaperone assistance during the folding process. Acetylcholinesterase (AChE)3 is an important component of the neuromuscular synapse where it rapidly terminates neurotransmission by hydrolyzing acetylcholine. Two separate genes encode the catalytic (AChE) and collagen tail (ColQ) subunits of the hetero-oligomeric collagen-tailed AChE forms expressed in skeletal muscle (for review, see Refs. 1-3). A single ColQAChE molecule consists of three catalytic AChE tetramers attached to a triple-helical collagen-like tail composed of three separate ColQ molecules. In some species and in some muscles, one (A4) or two (A8) tetramers of AChE attached to the triple helical ColQ subunits are observed. Each ColQ strand binds covalently to a tetramer of catalytic subunits, and each tetramer in turn consists of two dimers, with one dimer having its two C-terminal cysteine residues covalently linked to cysteine residues at the N terminus of ColQ. The other two catalytic subunits are also disulfide-linked to each other through the same residues (4, 5). Although many aspects of ColQ-AChE structure, function, and localization have been elucidated (for review, see Refs. 1-3), how this enzyme is regulated by muscle activity remains incompletely understood.Physiologically the expression of the synaptic ColQ-AChE form is regulated by the incoming nerve and by depolarization of the cell membrane (6 -11). Paradoxically, the direction of the regulation appears to be species-specific (for review, see Ref.3). In rats, for example, muscle paralysis due to denervation or induced pharmacologically leads to dramatic decrease in AChE transcripts and their accumulation at junctional sarcoplasm (12-14). Avian systems behav...

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Section: The Levels Of Endoplasmic Reticulum Molecular Chaperones Pdimentioning

confidence: 99%

Limiting Role of Protein Disulfide Isomerase in the Expression of Collagen-tailed Acetylcholinesterase Forms in Muscle

Ruiz¹,

Rotundo

2009

Journal of Biological Chemistry

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“…Two chaperones that may potentially play roles in a7-nAChR surface expression are calnexin and 14-3-3eta. Both can bind a subunits alone (a1 in muscle, and a4 in a4b2-nAChR, respectively), suggesting they may also bind the a7 subunit, and both can contribute to nAChR surface expression (Keller et al 1996;Chang et al 1997;Jeanclos et al 2001). Surface expression of a7-nAChR (but not of a4b2-nAChR) in transfected Xenopus oocytes has been shown to depend on the chaperone CyPA (Helekar et al 1994;Helekar and Patrick 1997).…”

Section: Discussionmentioning

confidence: 99%

Regulation by cycloheximide and lowered temperature of cell‐surface α7‐nicotinic acetylcholine receptor expression on transfected SH‐EP1 cells

Schroeder

Zhao

et al. 2003

Journal of Neurochemistry

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Heterologous expression of functional, nicotinic acetylcholine receptors (nAChR) in mammalian cells has been difficult to achieve or optimize, even for nAChR containing only one kind of subunit. In this study, we determined effects of lowered temperature or of exposure to the protein synthesis inhibitor cycloheximide (CHX) on cell surface expression of homomeric a7-nAChR in transfected SH-EP1 human epithelial cells. We found that incubation of cells for 2 days at 25°C or in the presence of 0.5-2 lg/mL of CHX caused four-or eight-fold increases, respectively, in surface binding sites for 125 I-labeled a-bungarotoxin (I-Bgt). These increases were accompanied by increases in peak whole-cell current responses to nicotinic agonists. Either treatment lowered protein synthesis and cell proliferation, but experiments using puromycin indicated that a reduction in protein synthesis or cell proliferation per se was not sufficient to increase surface binding. I-Bgt binding to whole-cell membrane pools increased in response to either treatment, suggesting that the increase in surface binding was due, at least in part, to an increase in intracellular receptor levels. The cyclophilin inhibitor cyclosporin A reduced surface expression in untreated as well as CHX-or 25°C-treated cells.The results suggest practical means for increasing cell surface and functional expression of a7-nAChR. Although these effects are not simply due to protein synthesis inhibition or reduced cell proliferation, they do involve an increase in intracellular receptor pool size.

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“…Studies in transfected cells have helped in characterizing the interaction of nAChRs with chaperone proteins such as BiP [277,278] and calnexin [279][280][281]. Such studies have also helped to reveal the role of nAChR-interacting proteins such as 14-3-3 [282,283] and VILIP-1 [284] in regulating cell-surface expression of α4β2 nAChRs.…”

Section: Co-expression With Chaperones and Interacting Proteinsmentioning

confidence: 99%

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

Millar

2009

Biochemical Pharmacology

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Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop family of neurotransmitter-gated ion channels, a family that also includes receptors for γ-aminobutyric acid, glycine and 5-hydroxytryptamine. In humans, nAChRs have been implicated in several neurological and psychiatric disorders and are major targets for pharmaceutical drug discovery. In addition, nAChRs are important targets for neuroactive pesticides in insects and in other invertebrates. Historically, nAChRs have been one of the most intensively studied families of neurotransmitter receptors. They were the first neurotransmitter receptors to be biochemically purified and the first to be characterized by molecular cloning and heterologous expression. Although much has been learnt from studies of native nAChRs, the expression of recombinant nAChRs has provided dramatic advances in the characterization of these important receptors. This review will provide a brief history of the characterization of nAChRs by heterologous expression. It will focus, in particular, upon studies of recombinant nAChRs, work that has been conducted by many hundreds of scientists during a period of almost 30 years since the molecular cloning of nAChR subunits in the early 1980's.

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Calnexin-dependent Enhancement of Nicotinic Acetylcholine Receptor Assembly and Surface Expression

Cited by 73 publications

References 45 publications

Limiting Role of Protein Disulfide Isomerase in the Expression of Collagen-tailed Acetylcholinesterase Forms in Muscle

Limiting Role of Protein Disulfide Isomerase in the Expression of Collagen-tailed Acetylcholinesterase Forms in Muscle

Regulation by cycloheximide and lowered temperature of cell‐surface α7‐nicotinic acetylcholine receptor expression on transfected SH‐EP1 cells

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

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