Conformational Changes Induced in the Endoplasmic Reticulum Luminal Domain of Calnexin by Mg-ATP and Ca2+

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...

Section: Protein Disulfide Isomerase and Qcolq Interact Synergisticalmentioning

confidence: 83%

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

Ruiz¹,

Rotundo

2009

“…Although the identity of the interacting cellular machinery and its distribution among different types of cells remain to be established, ER chaperones such as calnexin (a type I integral membrane protein) (39) have been shown to regulate receptor subunit folding, assembly, and half-life and are thus potential components of the yet-to-be-identified protein complex (40,41). It is worth noting that Cys-loop superfamily receptors are present not only in neurons but also in myocytes, epithelial cells, and lymphocytes (42,43).…”

Section: Mutation Of the Conserved Aspartate Residue Compromised Pentmentioning

confidence: 99%

A Conserved Cys-loop Receptor Aspartate Residue in the M3-M4 Cytoplasmic Loop Is Required for GABAA Receptor Assembly

Botzolakis

Tang

et al. 2008

Members of the Cys-loop superfamily of ligand-gated ion channels, which mediate fast synaptic transmission in the nervous system, are assembled as heteropentamers from a large repertoire of neuronal subunits. Although several motifs in subunit N-terminal domains are known to be important for subunit assembly, increasing evidence points toward a role for C-terminal domains. Using a combination of flow cytometry, patch clamp recording, endoglycosidase H digestion, brefeldin A treatment, and analytic centrifugation, we identified a highly conserved aspartate residue at the boundary of the M3-M4 loop and the M4 domain that was required for binary and ternary ␥-aminobutyric acid type A receptor surface expression. Mutation of this residue caused mutant and partnering subunits to be retained in the endoplasmic reticulum, reflecting impaired forward trafficking. Interestingly although mutant and partnering wild type subunits could be coimmunoprecipitated, analytic centrifugation studies demonstrated decreased formation of pentameric receptors, suggesting that this residue played an important role in later steps of subunit oligomerization. We thus conclude that C-terminal motifs are also important determinants of Cys-loop receptor assembly.The Cys-loop superfamily of ligand-gated ion channels, which includes ␥-aminobutyric acid type A (GABA A ) 2 and type C (GABA C ), nicotinic acetylcholine, glycine, and 5-hydroxytryptamine type 3 receptors, mediates fast synaptic transmission in the nervous system. Mutations that alter Cys-loop receptor surface density by affecting receptor biogenesis have been associated with idiopathic generalized epilepsies (1-4), congenital myasthenic syndromes (5), and psychiatric disorders (6). Unfortunately because the structural and cellular determinants of receptor biogenesis are poorly understood, development of effective treatment strategies remains a significant challenge.A wealth of evidence suggests that Cys-loop receptors are assembled as heteropentamers from a large repertoire of neuronal subunits (7-10). Subunits share a similar topology that includes an extracellular N-terminal domain, four transmembrane domains, three loops including a large cytoplasmic loop, and a variable length extracellular C-terminal tail (7, 11). Receptor assembly is thought to occur in the endoplasmic reticulum (ER) following glycosylation and folding of de novo synthesized subunits (12-15). Assembly is closely monitored by ER quality control machinery, and consequently subunits that fail to assemble properly are retained and degraded (15)(16)(17). Although N-terminal motifs are known to be important for subunit assembly (18,19), recent studies in nicotinic acetylcholine receptors (nAChRs) suggest that C-terminal motifs may also play a role (20).GABA A receptors are the most abundant Cys-loop receptor in the mammalian brain and are responsible for the majority of fast inhibitory neurotransmission. Like other Cys-loop superfamily receptors, they are pentamers assembled from combinations of 16 subunit subtypes ...

“…The ability of CN to bind to the protein N-linked glycan is lost with DTT treatment (38), likely via reduction of a disulfide bond within the lectin-binding domain (36). To test whether DTT exposure affects associations between CN and the subunits, cells transiently expressing each AChR subunit along with CN-HA were treated with 5 mM DTT.…”

Section: N-linked Glycans and Nicotinic Receptor Assemblymentioning

confidence: 99%

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

Wanamaker

Green

2005

We investigated how asparagine (N)-linked glycosylation affects assembly of acetylcholine receptors (AChRs) in the endoplasmic reticulum (ER). Block of N-linked glycosylation inhibited AChR assembly whereas block of glucose trimming partially blocked assembly at the late stages. Removal of each of seven glycans had a distinct effect on AChR assembly, ranging from no effect to total loss of assembly. Because the chaperone calnexin (CN) associates with N-linked glycans, we examined CN interactions with AChR subunits. CN rapidly associates with 50% or more of newly synthesized AChR subunits, but not with subunits after maturation. Block of N-linked glycosylation or trimming did not alter CN-AChR subunit associations nor did subunit mutations prevent N-linked glycosylation. Additionally, CN associations with subunits lacking N-linked glycans occurred without subunit aggregation or misfolding. Our data indicate that CN associates with AChR subunits without N-linked glycan interactions. Furthermore, CN-subunit associations only occur early in AChR assembly and have no role in events later that require N-linked glycosylation. The endoplasmic reticulum (ER)2 lumen is specialized for protein folding and assembly. Resident chaperone proteins facilitate protein folding while processing enzymes catalyze post-translational protein processing events such as the addition of asparagine (N)-linked glycans and disulfide bond formation. The role of N-linked glycans in protein folding, assembly, and function is not clearly defined. Prevention of N-linked glycosylation has varied effects among proteins and depends on many different factors (1). One function of N-linked glycosylation is to mediate associations with certain ER-resident chaperones. Whereas most ER-resident chaperone proteins bind directly to polypeptide regions of proteins, calnexin (CN) and calreticulin (CRT) can bind to N-linked glycans of proteins via its lectin binding domain. Both specifically associate with monoglucosylated Glc 1 Man 9 GlcNAc 2 oligosaccharides transiently during glucose trimming of N-linked glycans in the ER (reviewed in Refs. 1-3). As a lectin, CN binds glycans to deliver proteins to other chaperones and processing enzymes. Recent studies suggest that CN is part of a larger complex that contains other components such as the thiol oxidoreductase ERp57 (reviewed in Ref. 4).Can CN bind its substrates through an N-linked glycosylation-independent mechanism? Several studies found that CN interactions with proteins occur after enzymatic removal of glycans (5-7) and that glycosylation or glucosidase inhibitors did not completely inhibit associations with CN (8 -13). Also, CN associates with proteins not N-linked glycosylated, such as the T-cell receptor ⑀-subunit (14), a mutant proteolipid protein (15) or proteins mutated to eliminate consensus N-linked glycosylation sites, such as the multidrug P-glycoprotein (16). Recent results using various CN deletion or point mutations found regions of CN that can prevent aggregation of non-glycosylated proteins...