N-Linked Glycosylation Is Required for Nicotinic Receptor Assembly but Not for Subunit Associations with Calnexin

Wanamaker, Christian; Green, William N.

doi:10.1074/jbc.m501813200

Cited by 39 publications

(41 citation statements)

References 57 publications

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“…AChR Subunits Associate with ERp57-We previously had characterized interactions between CN and nAChR subunits and found that CN rapidly associates with each nAChR subunit, but the interactions did not depend on subunit N-linked glycosylation (7). In this study, we performed a similar set of experiments to characterize interactions between ERp57 and nAChR subunits.…”

Section: Resultsmentioning

confidence: 88%

“…directly with N-linked glycan (4) and/or with polypeptide domains (5)(6)(7). CN is found in a complex with ERp57, a member of the protein-disulfide isomerase superfamily that catalyzes the formation of intramolecular disulfide bonds in nascent proteins.…”

mentioning

confidence: 99%

“…All three chaperones have been found to associate with unassembled subunits. CN associates with all four unassembled subunits (7,24,27), ERp57 associates with unassembled ␦ subunits (28), and BiP associates with unassembled ␣ subunits (29 -31). To examine the role of CN, ERp57, and BiP during the assembly of the AChR subunits, we used the AChR subunits from Torpedo californica.…”

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confidence: 99%

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Endoplasmic Reticulum Chaperones Stabilize Nicotinic Receptor Subunits and Regulate Receptor Assembly

Wanamaker

Green

2007

Journal of Biological Chemistry

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We examined interactions between the endoplasmic reticulum (ER) chaperones calnexin (CN), ERp57, and immunological heavy chain-binding protein (BiP) and nicotinic acetylcholine receptor (nAChR) subunits. The three chaperones rapidly associate with newly synthesized nAChR subunits. Interactions between nAChR subunits and ERp57 occur via transient intermolecular disulfide bonds and do not require subunit N-linked glycosylation. The associations of ERp57 or CN with AChR subunits are long lived and prolong subunit lifetime ϳ10-fold. Coexpression of CN or ERp57 alone does not affect nAChR assembly or trafficking, but together they cause a significant decrease in nAChR expression and assembly. In contrast, associations with BiP are shorter lived and do not alter nAChR expression and assembly. However, a mutated BiP that slows its dissociation significantly increases its associations and decreases nAChR expression and assembly. Our results suggest that interactions with the chaperones regulate the levels of nAChRs assembled in the ER by stabilizing and sequestering subunits during assembly. The endoplasmic reticulum (ER)2 is highly specialized to promote and regulate the synthesis and maturation of membrane and secreted proteins. These processes are dependent on quality ER control mechanisms that identify and degrade misfolded proteins. Components of the ER quality control machinery are molecular chaperones (for reviews, see Refs. 1 and 2). Recent studies have identified protein domains and processing events that mediate interactions between ER chaperones and their substrates (3). Less is known about chaperone-substrate dynamics after the interaction is established.Here, we examine interactions between nicotinic acetylcholine receptor (nAChR) subunits and the ER chaperones, calnexin (CN), ERp57, and immunological heavy chain-binding protein (BiP). CN is a type I membrane protein that interacts directly with N-linked glycan (4) and/or with polypeptide domains (5-7). CN is found in a complex with ERp57, a member of the protein-disulfide isomerase superfamily that catalyzes the formation of intramolecular disulfide bonds in nascent proteins. ERp57 has been shown to specifically recognize only trimmed glycoproteins (8 -10). However, the lectin specificity of ERp57 may be a result of its association with CN rather than an intrinsic ERp57 lectin domain (11-13), which suggests cooperativity between CN and ERp57. Meanwhile, BiP function occurs via ATP-driven cycles of binding to and release of substrate proteins, which appears to promote protein folding by maintaining a state competent for folding by preventing aggregation (14). Release of substrate requires ATP hydrolysis by the BiP ATPase (15), and ATPase mutations block substrate release (16). BiP plays a variety of additional roles in the ER, including the translocation of newly synthesized proteins across the ER membrane (17, 18) and a role in proteasome degradation (19,20).In this study, we have characterized how interactions between the ER chaperones and muscle nAChR su...

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Section: Resultsmentioning

confidence: 88%

mentioning

confidence: 99%

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confidence: 99%

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Endoplasmic Reticulum Chaperones Stabilize Nicotinic Receptor Subunits and Regulate Receptor Assembly

Wanamaker

Green

2007

Journal of Biological Chemistry

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“…The observed lectinindependent binding of some substrates to Cnx or Crt could simply be due to their inclusion in protein aggregates. This may be the case in some instances (Cannon et al, 1996) but the possibility has been rigorously excluded in others, in which aggregates were tested for by density gradient centrifugation (Danilczyk and Williams, 2001;Wanamaker and Green, 2005).…”

Section: Mechanisms Of Action: Lectin-only or Dual-binding?mentioning

confidence: 99%

Beyond lectins: the calnexin/calreticulin chaperone system of the endoplasmic reticulum

Williams

2006

Journal of Cell Science

424

359

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The structure of the ER-luminal portion of Cnx, solved by Xray crystallography (Schrag et al., 2001), reveals two domains: a globular ␤-sandwich domain that resembles legume lectins; and an extended 140 Å arm domain consisting of two ␤-strands folded in a hairpin configuration ( Fig. 2A). Each ␤-strand is composed of four tandemly repeated, proline-rich repeats, which is why the extended arm is frequently referred to as the P domain. For Crt, only the arm domain structure has been solved by NMR methods. It is similar to that of Cnx but shorter,

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“…AChR subunits enter the ER cotranslationally in an unfolded state via the Sec61 translocon. Transient interactions with ER resident components of the biosynthetic machinery, such as BiP (13,14), calnexin (15,16), and ERp57 (17), then influence the folding process and protect immature subunits from degradation. Folding subunits are exposed to a variety of enzymes that catalyze posttranslational modifications (including glycosylation and palmitoylation) and regulate protein expression (18)(19)(20).…”

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confidence: 99%

Biosynthesis of ionotropic acetylcholine receptors requires the evolutionarily conserved ER membrane complex

Richard

Boulin

Robert

et al. 2013

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

105

146

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The number of nicotinic acetylcholine receptors (AChRs) present in the plasma membrane of muscle and neuronal cells is limited by the assembly of individual subunits into mature pentameric receptors. This process is usually inefficient, and a large number of the synthesized subunits are degraded by endoplasmic reticulum (ER)-associated degradation. To identify cellular factors required for the synthesis of AChRs, we performed a genetic screen in the nematode Caenorhabditis elegans for mutants with decreased sensitivity to the cholinergic agonist levamisole. We isolated a partial loss-of-function allele of ER membrane protein complex-6 (emc-6), a previously uncharacterized gene in C. elegans. emc-6 encodes an evolutionarily conserved 111-aa protein with two predicted transmembrane domains. EMC-6 is ubiquitously expressed and localizes to the ER. Partial inhibition of EMC-6 caused decreased expression of heteromeric levamisole-sensitive AChRs by destabilizing unassembled subunits in the ER. Inhibition of emc-6 also reduced the expression of homomeric nicotine-sensitive AChRs and GABA A receptors in C. elegans muscle cells. emc-6 is orthologous to the yeast and human EMC6 genes that code for a component of the recently identified ER membrane complex (EMC). Our data suggest this complex is required for protein folding and is connected to ER-associated degradation. We demonstrated that inactivation of additional EMC members in C. elegans also impaired AChR synthesis and induced the unfolded protein response. These results suggest that the EMC is a component of the ER folding machinery. AChRs might provide a valuable proxy to decipher the function of the EMC further.

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N-Linked Glycosylation Is Required for Nicotinic Receptor Assembly but Not for Subunit Associations with Calnexin

Cited by 39 publications

References 57 publications

Endoplasmic Reticulum Chaperones Stabilize Nicotinic Receptor Subunits and Regulate Receptor Assembly

Endoplasmic Reticulum Chaperones Stabilize Nicotinic Receptor Subunits and Regulate Receptor Assembly

Beyond lectins: the calnexin/calreticulin chaperone system of the endoplasmic reticulum

Biosynthesis of ionotropic acetylcholine receptors requires the evolutionarily conserved ER membrane complex

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