Regulation of nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and cAMP

K. Miles, D. T. Anthony, L. L. Rubin, P. Greengard, Richard L Huganir

Research output: Contribution to journalArticle

Abstract

The nicotinic acetylcholine receptor (Ac-ChoR) 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 μM in the presence of 35 μM 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.

Original languageEnglish (US)
Pages (from-to)6591-6595
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume84
Issue number18
StatePublished - 1987
Externally publishedYes

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Skeletal Muscle Fibers
Nicotinic Receptors
Colforsin
Phosphorylation
Phosphopeptides
4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone
Phosphodiesterase Inhibitors
Cyclic AMP-Dependent Protein Kinases
Affinity Chromatography
Adenylyl Cyclases
Acetylcholine
Chromatography
Cell Culture Techniques
Phosphates
Muscles

ASJC Scopus subject areas

  • General
  • Genetics

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Regulation of nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and cAMP. / Miles, K.; Anthony, D. T.; Rubin, L. L.; Greengard, P.; Huganir, Richard L.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 84, No. 18, 1987, p. 6591-6595.

Research output: Contribution to journalArticle

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T1 - Regulation of nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and cAMP

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AU - Anthony, D. T.

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AU - Huganir, Richard L

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