Arrest of Subunit Folding and Assembly of Nicotinic Acetylcholine Receptors in Cultured Muscle Cells by Dithiothreitol

Gelman, Marina S.; Prives, Joav

doi:10.1074/jbc.271.18.10709

Cited by 23 publications

(16 citation statements)

References 46 publications

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“…2B). This result is at odds with a recent study (19), which suggested that 5 mM DTT completely blocks AChR subunit assembly. There (30), the rat brain nAChR ␣2, ␤2 (31) and ␣7 (32) subunits, the C. elegans nAChR subunit deg-3 (33), the calf brain GABA A receptor ␣1 and ␤1 subunits (34,35), the rat brain glycine receptor ␣1 subunit (36), the mouse 5HT3 receptor subunit (37), and the rat brain glutamate receptor subunit Glu R1 (11).…”

Section: Reduction Of Disulfide Bonds Alters Subunit Assembly-contrasting

confidence: 56%

The Role of the Cystine Loop in Acetylcholine Receptor Assembly

Green

Wanamaker

1997

Journal of Biological Chemistry

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Nicotinic acetylcholine receptors (AChRs) are composed of ␣, ␤, ␥, and ␦ subunits, assembled into ␣ 2 ␤␥␦ pentamers. A highly conserved feature of ionotropic neurotransmitter receptors, such as AChRs, is a 15-amino acid cystine "loop." We find that an intact cystine loop is necessary for complete AChR assembly. By preventing formation of the loop with 5 mM dithiothreitol, AChR subunits assemble into ␣␤␥ trimers, but the subsequent steps in assembly are blocked. When ␣ subunit loop cysteines are mutated to serines, assembly is blocked at the same step as with dithiothreitol. In contrast, when ␤ subunit loop cysteines are mutated to serines, assembly is blocked at a later step, i.e. after assembly of ␣␤␥␦ tetramers and before the addition of the second ␣ subunit. After formation of the cystine loop, the ␣ subunit undergoes a conformational change, which buries the loop. This conformational change is concurrent with the step in assembly blocked by removal of the disulfide bond of the cystine loop, i.e. after assembly of ␣␤␥ trimers and before the addition of the ␦ subunit. The data indicate that the ␣ subunit conformational change involving the cystine loop is key to a series of folding events that allow the addition of unassembled subunits.The molecular events involved in subunit folding and assembly of large oligomeric proteins remain largely uncharacterized. The subunit folding and oligomerization events that take place during the assembly of ion channels are particularly complex, since the finished product requires the correct oligomeric arrangement and subunit stoichiometry for proper function (1). In terms of their structure, the best characterized ion channels are the muscle-type nicotinic acetylcholine receptors (AChRs), 1 which are the neurotransmitter receptors responsible for rapid signaling between motor neurons and skeletal muscle. Muscle-type AChRs are composed of four distinct, homologous subunits, ␣, ␤, ␥, and ␦, which assemble into pentamers with the subunit stoichiometry of ␣ 2 ␤␥␦.Although AChR assembly is a slow process that takes ϳ2 h to complete (2), assembly intermediates have been difficult to isolate. A number of laboratories turned to expression of less than the full complement of subunits in different heterologous expression systems to isolate assembly intermediates (3-6).Based on their findings, the "heterodimer" model was proposed, where the ␣ subunit must first fold or "mature," as assayed by the formation of the ␣-bungarotoxin (BuTx) binding site and antigenic epitopes, before assembling with other subunits. The mature ␣ subunit assembles with ␥ or ␦ subunits in parallel to form ␣␥ and ␣␦ heterodimers, and the heterodimers associate together and with ␤ subunits to form ␣ 2 ␤␥␦ pentamers.We have developed techniques that have allowed isolation of assembly intermediates in cells stably expressing all four AChR subunits (7, 8) and have obtained results at odds with the heterodimer model. Instead of heterodimers, two partially assembled complexes, ␣␤␥ trimers and ␣␤␥␦ tetramers, were isol...

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Section: Reduction Of Disulfide Bonds Alters Subunit Assembly-contrasting

confidence: 56%

The Role of the Cystine Loop in Acetylcholine Receptor Assembly

Green

Wanamaker

1997

Journal of Biological Chemistry

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“…Other transfected AChR subunits were also found to interact with calnexin in HEK 293 cells (25,26). In muscle cells, the timing of this interaction appears to be precisely regulated, with binding occurring immediately upon subunit translation and dissociation taking place prior to AChR assembly (24,27). This time course is consistent with the possibility that calnexin assists AChR assembly by facilitating the folding of nascent subunits into an assembly-competent conformation.…”

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confidence: 65%

“…Using cultured muscle cells, we have recently shown that AChR assembly can be blocked as a result of impaired subunit folding in the presence of dithiothreitol, a reducing agent which prevents disulfide bond formation on nascent subunits (27). We have now found that AChR assembly is also impaired upon inhibition of another post-translational modification of nascent AChR subunits, the trimming of glucose residues on N-linked oligosaccharides, induced by CAS.…”

Section: Discussionmentioning

confidence: 53%

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

Chang

Gelman

Prives

1997

Journal of Biological Chemistry

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The muscle-type nicotinic acetylcholine receptor (AChR) 2 is a pentameric membrane ion channel assembled in the endoplasmic reticulum from four homologous subunits by mechanisms that are insufficiently understood. Nascent AChR subunits were recently found to form complexes with the endoplasmic reticulum-resident molecular chaperone calnexin. To determine the contribution of this interaction to AChR assembly and surface expression, we have now used transient transfection of mouse AChR subunits and calnexin into nonmuscle cells. Co-transfection of calnexin along with AChR subunits into COS and HEK 293 cells was found to enhance AChR subunit folding and assembly, and to decrease degradation rates of newly synthesized AChR ␣-subunits, resulting in elevated surface expression of assembled AChR. Moreover, inhibition of the interaction between endogenous calnexin and AChR by castanospermine resulted in decreased AChR subunit folding, assembly, and surface expression in muscle and HEK 293 cells. Together, these findings provide evidence that calnexin directly contributes to AChR biogenesis by promoting subunit folding and assembly.

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“…1B). In this context, it is of relevance to mention that the membranepermeating reductants generate an intracellular reducing environment that affects protein folding and, in particular, disulfide bond formation (18,19). Considering that ArcB possesses a cytoplasm-located signal reception site (6), it is activated only by Four parallel cultures of strain ECL5203 (6) and its isogenic ECL5204 (⌬arcB) (6) were grown aerobically in Luria-Bertani broth containing 0.1 M MOPS (pH 7.4) and 20 mM D-xylose.…”

Section: Effects Of Various Modifyingmentioning

confidence: 99%

Identification of a quinone-sensitive redox switch in the ArcB sensor kinase

Malpica

Franco

Rodríguez

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

262

254

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Escherichia coli senses and signals anoxic or low redox conditions in its growth environment by the Arc two-component system. Under anaerobic conditions, the ArcB sensor kinase autophosphorylates and transphosphorylates ArcA, a global transcriptional regulator that controls the expression of numerous operons involved in respiratory or fermentative metabolism. Under aerobic conditions, the kinase activity of ArcB is inhibited by the quinone electron carriers that act as direct negative signals. Here, we show that the molecular mechanism of kinase silencing involves the oxidation of two cytosol-located redox-active cysteine residues that participate in intermolecular disulfide bond formation, a reaction in which the quinones provide the source of oxidative power. Thus, a pivotal link in the Arc signal transduction pathway connecting the redox state of the quinone pool to the transcriptional apparatus is elucidated.T wo-component signal transduction systems are widespread in prokaryotes and play extensive roles in adaptation to environmental changes (1, 2). The Arc (anoxic redox control) two-component system is an important element in the complex transcriptional regulatory network that allows facultative anaerobic bacteria, such as Escherichia coli, to sense various respiratory growth conditions and adapt their gene expression accordingly (3). This system comprises the cytoplasmic response regulator ArcA and the transmembrane sensor kinase ArcB (4, 5). ArcA is a typical response regulator possessing an N-terminal receiver domain with a conserved Asp residue at position 54 and a C-terminal helix-turn-helix DNA binding domain. In contrast, ArcB is an unorthodox sensor kinase as manifested by its unusually elaborate architecture. As a sensor, ArcB is deviant because in contrast to typical sensor kinases that have a substantial periplasmic domain for environmental sensing, ArcB has a very short periplasmic sequence of only 16 amino acid residues delimited by two canonical transmembrane segments. Interestingly, the ArcB transmembrane domain (amino acids 22-77) does not directly participate in signal sensing but rather serves as an anchor that keeps the protein close to the source of the signal (6). As a kinase, ArcB is atypical because it contains three catalytic domains: an N-terminal transmitter domain with a conserved His-292 residue, a central receiver domain with a conserved Asp-576 residue, and a C-terminal phosphotransfer domain with a conserved His-717 residue (5, 7). Moreover, in the linker that is the region connecting the catalytic domains with the transmembrane domain, there are a putative leucine zipper (8) and a Per-Arnt-Sim (PAS) domain (9).Under reducing conditions, ArcB undergoes ATP-dependent autophosphorylation, a process shown to be enhanced by certain anaerobic metabolites such as D-lactate, acetate, and pyruvate (10, 11), and transphosphorylates ArcA via a His-292 3 Asp-576 3 His-717 3 Asp-54 phosphorelay (12, 13). Phosphorylated ArcA (ArcA-P), in turn, represses the expression of many operons invo...

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Arrest of Subunit Folding and Assembly of Nicotinic Acetylcholine Receptors in Cultured Muscle Cells by Dithiothreitol

Cited by 23 publications

References 46 publications

The Role of the Cystine Loop in Acetylcholine Receptor Assembly

The Role of the Cystine Loop in Acetylcholine Receptor Assembly

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

Identification of a quinone-sensitive redox switch in the ArcB sensor kinase

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