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 beta and delta 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 delta subunit 20-fold over basal phosphorylation and induced phosphorylation of the alpha subunit. The effect of forskolin was dose dependent with a half-maximal response at 8 microM in the presence of 35 microM Ro 20-1724, a phosphodiesterase inhibitor. Stimulation of delta subunit phosphorylation was almost maximal within 5 min, whereas stimulation of alpha 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 delta 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 delta subunit increases the rate of AcChoR desensitization in rat myotubes.
Miles
K
K
Anthony
D T
DT
Rubin
L L
LL
Greengard
P
P
Huganir
R L
RL
D000818:Animals; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000494:pharmacology*; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism*; D009469:Neuromuscular Junction / Q000502:physiology; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011978:Receptors, Nicotinic / Q000378:metabolism*
D046911:Macromolecular Substances; D008565:Membrane Proteins; D010750:Phosphoproteins; D011978:Receptors, Nicotinic; D005576:Colforsin; D000242:Cyclic AMP; D011494:Protein Kinases