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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 approximately 10-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.
Highlights
We examine interactions between nicotinic acetylcholine receptor subunits and the endoplasmic reticulum (ER) chaperones, calnexin (CN), ERp57, and immunological heavy chain-binding protein (BiP)
CN rapidly associates with each nicotinic acetylcholine receptor (nAChR) subunit, but the interactions did not depend on subunit
To determine the percentage of nAChR subunit complexed with ERp57-HA, the amount of labeled subunit co-immunoprecipitated with an anti-HA Ab (Fig. 1B, lanes 2, 4, 6, and 8) was compared with the total amount of subunit synthesis as assayed by immunoprecipitation with subunit-specific Abs (Fig. 1B, lanes 1, 3, 5, and 7)
Summary
Tissue Culture—The primary cell line used for transient transfection experiments was the tsA201 cell line, a human embryonic kidney cell line derived from HEK293 cells [33]. The first round of PCR involved using a standard pRBG4 vector forward primer or reverse primer and a complementary ERp57-specific primer that encoded 1) 21–24 base pairs of the HA epitope tag and 2) 20 –22 base pairs of ERp57. Transient Transfection—The chaperones, Torpedo and mouse AChR subunit cDNAs, were transiently transfected into 6-cm cultures of tsA201 cells [33] using a calcium phosphate method [35]. When testing for the effect of a chaperone on mouse AChR expression, we transfected the cells with 5 g of ␣ and 2.5 g each of , ⑀ (or ␥), and ␦ subunit along with 10 g of CN-HA, ERp57-HA, or empty vector or 5 g each of CN-HA and ERp57-HA. Band intensities were quantified by scanning 3–5 exposures to ensure the linearity of the band
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