Analysis of early events in acetylcholine receptor assembly.

Paulson, Henry L.; Ross, Anthony F.; Green, William N.; Claudio, Toni

doi:10.1083/jcb.113.6.1371

Cited by 50 publications

(41 citation statements)

References 45 publications

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“…In the case of mutant CFTR protein, the efficiency of cellular membrane integration declines to ,25% that of the wild-type protein (Kopito, 1999). Similar observations have been made for a number of other integral membrane proteins, such as epithelial sodium channel (ENaC), band 3 anion exchanger (SLC4A1) and nicotinic acetylcholine receptor (CHRNA) (Paulson et al, 1991;Rajan et al, 2001;Valentijn et al, 1998). Protein folding and stability studies have demonstrated that a single amino acid change is sufficient to reduce the folding efficiency in a protein from 50% at body temperature to below 10% (Guerois et al, 2002).…”

Section: Introductionsupporting

confidence: 53%

Disease-associated missense mutations in bestrophin-1 affect cellular trafficking and anion conductance

Milenkovic

Röhrl

Weber

et al. 2011

Journal of Cell Science

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SummaryBestrophin-1, an integral membrane protein encoded by the BEST1 gene, is localized predominantly to the basolateral membrane of the retinal pigment epithelium. Mutations in the BEST1 gene have been associated with Best vitelliforme macular dystrophy (BMD), a central retinopathy with autosomal dominant inheritance and variable penetrance. Over 120 disease-causing mutations are known, the majority of which result in amino acid substitutions within four mutational hot-spot regions in the highly conserved N-terminal half of the protein. Although initially thought to impair Cl -channel function, the molecular pathology of BEST1 mutations is still controversial. We have analyzed the subcellular localization of 13 disease-associated BEST1 mutant proteins in polarized MDCK II cells, an established model of apical to basolateral protein sorting. Immunostaining demonstrated that nine of the 13 mutant proteins failed to integrate into the cell membrane. The defective proteins were predominantly retained in the cytoplasm, whereas wild-type bestrophin-1 revealed cell membrane localization. Functional analysis of I -fluxes in HEK-293 cells showed that all mutants exhibited a significant reduction in anion conductance. Our data indicate that defective intracellular trafficking could be a common cause of BMD accompanied by impaired anion conductance, representing a loss of anion channel function that is probably due to mistargeting of mutant protein.

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Section: Introductionsupporting

confidence: 53%

Disease-associated missense mutations in bestrophin-1 affect cellular trafficking and anion conductance

Milenkovic

Röhrl

Weber

et al. 2011

Journal of Cell Science

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“…5, B and D). The subunits migrated broadly across the gradients, as previously observed for unassembled AChR subunits (40), appearing in fractions 3-4 where subunit monomers are expected to migrate and continuing to the bottom fractions. The migration of the subunits is consistent with the formation of heterogeneous homo-oligomers of the subunits and/or associations with chaperone proteins such as CN (see also Ref.…”

Section: N-linked Glycans and Nicotinic Receptor Assemblymentioning

confidence: 49%

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

Wanamaker

Green

2005

Journal of Biological Chemistry

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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...

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“…The nascent subunit polypeptide subsequently undergoes cleavage of its signal sequence, oxidation of its disulfide bonds (Mishina et al, 1985;Blount and Merlie, 1990), and N-glycosylation of specific residues (Merlie et al, 1982;Smith et al, 1987;Blount and Merlie 1988). Chaperones such as binding protein BiP (Blount and Merlie, 1991;Paulson et al, 1991;Forsayeth et al, 1992) and calnexin (Gelman et al, 1995;Keller et al, 1996Keller et al, , 1998 promote proper folding and maturation of AChR subunits. Unassembled or misfolded AChR subunits are then targeted for degradation by the ER-associated proteasomal degradation machinery (Wanamaker et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Structural Determinants of α4β2 Nicotinic Acetylcholine Receptor Trafficking

Ren¹,

Cheng²,

Treuil³

et al. 2005

J. Neurosci.

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The structural determinants of nicotinic acetylcholine receptor (AChR) trafficking have yet to be fully elucidated. Hydrophobic residues occur within short motifs important for endoplasmic reticulum (ER) export or endocytotic trafficking. Hence, we tested whether highly conserved hydrophobic residues, primarily leucines, in the cytoplasmic domain of the ␣4␤2 AChR subunits were required for cell surface expression of ␣4␤2 AChRs. Mutation of F350, L351, L357, and L358 to alanine in the ␣4 AChR subunit attenuates cell surface expression of mutant ␣4␤2 AChRs. Mutation of F342, L343, L349, and L350 to alanine at homologous positions in the ␤2 AChR subunit abolishes cell surface expression of mutant ␣4␤2 AChRs. The hydrophobic nature of the leucine residue is a primary determinant of its function because mutation of L343 to another hydrophobic amino acid, phenylalanine, in the ␤2 AChR subunit only poorly inhibits trafficking of mutant ␣4␤2 AChR to the cell surface. All mutant ␣4␤2 AChRs exhibit high-affinity binding for [ 3 H]epibatidine. In both tsA201 cells and differentiated SH-SY5Y neural cells, wild-type ␣4␤2 AChRs colocalize with the Golgi marker giantin, whereas mutant ␣4␤2 AChRs fail to do so. The striking difference between mutant ␣4 versus mutant ␤2 AChR subunits on cell surface expression of mutant ␣4␤2 AChRs points to a cooperative or regulatory role for the ␣4 AChR subunit and an obligatory role for the ␤2 AChR subunit in ER export. Collectively, our results identify, for the first time, residues within AChR subunits that are essential structural determinants of ␣4␤2 AChR ER export.

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Analysis of early events in acetylcholine receptor assembly.

Cited by 50 publications

References 45 publications

Disease-associated missense mutations in bestrophin-1 affect cellular trafficking and anion conductance

Disease-associated missense mutations in bestrophin-1 affect cellular trafficking and anion conductance

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

Structural Determinants of α4β2 Nicotinic Acetylcholine Receptor Trafficking

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