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Regulation of Nicotinic Acetylcholine Receptor Phosphorylation in Rat Myotubes by Forskolin and cAMP

The nicotinic acetylcholine receptor (AcChoR) from rat myotubes prelabeled in culture with [32P]orthophosphate was isolated by acetylcholine affinity chromatography followed by immunoaffinity chromatography. Under basal conditions, the nicotinic AcChoR was shown to be phosphorylated in situ on the β...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1987-09, Vol.84 (18), p.6591-6595
Main Authors: Miles, Kathryn, Anthony, Donna T., Rubin, Lee L., Greengard, Paul, Huganir, Richard L.
Format: Article
Language:English
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Summary:The nicotinic acetylcholine receptor (AcChoR) from rat myotubes prelabeled in culture with [32P]orthophosphate was isolated by acetylcholine affinity chromatography followed by immunoaffinity chromatography. Under basal conditions, the nicotinic AcChoR was shown to be phosphorylated in situ on the β and δ subunits. Regulation of AcChoR phosphorylation by cAMP-dependent protein kinase was explored by the addition of forskolin or cAMP analogues to prelabeled cell cultures. Forskolin, an activator of adenylate cyclase, stimulated the phosphorylation of the δ subunit 20-fold over basal phosphorylation and induced phosphorylation of the α subunit. The effect of forskolin was dose dependent with a half-maximal response at 8 μ M in the presence of 35 μ M Ro 20-1724, a phosphodiesterase inhibitor. Stimulation of δ subunit phosphorylation was almost maximal within 5 min, whereas stimulation of α subunit phosphorylation was not maximal until 45 min after forskolin treatment. Stimulation of AcChoR phosphorylation by 8-benzylthioadenosine 3′,5′-cyclic monophosphate was identical to that obtained by forskolin. Two-dimensional thermolytic phosphopeptide maps of the δ subunit revealed a single major phosphopeptide. These results correlate closely with the observed effects of forskolin on AcChoR desensitization in muscle and suggest that cAMP-dependent phosphorylation of the δ subunit increases the rate of AcChoR desensitization in rat myotubes.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.84.18.6591