Endoplasmic Reticulum Chaperones Stabilize Nicotinic Receptor Subunits and Regulate Receptor Assembly

Abstract

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...

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

“…The calnexin and ERp57 ER-resident component of the biosynthetic machinery also form transient complexes with newly synthesized AChRs before full assembly occurs (17). The association of AChR subunits with ER proteins would decrease the rate of ERAD-dependent subunit degradation (17).…”

“…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).…”

Sorting receptor Rer1 controls surface expression of muscle acetylcholine receptors by ER retention of unassembled α-subunits

“…Newly synthesized subunits interact with the general folding machinery including calnexin and BIP (16). In Caenorhabditis elegans, three genes are essential for functional reconstitution of the levamisole-sensitive AChR (L-AChR) (17).…”

Emerging role of ER quality control in plant cell signal perception

“…In human cells, the chaperones in the ER, like CRT, ERP57 and BiP, associate with unassembled subunits or folding intermediates to regulate subunit stability, assembly and expression of receptor complex (Wanamaker and Green, 2007). The ER chaperones are involved in different biological processes with specific substrates to control the response to extracellular stimuli.…”

“…It is composed of 2α,β,γ and δ subunits. Evidence has shown that BiP, ERp57 and CNX associate with newly synthesized AchR subunits by stabilizing and sequestering them to regulate the level of assembled AchR in the ER (Wanamaker and Green, 2007). A conserved motif in the transmembrane domain of the subunits functions in the ER retention of the receptor.…”