Mapping of the cAMP-dependent phosphorylation sites on the acetylcholine receptor.
We have synthesized a tetradecapeptide corresponding to residues 354-367 of the delta-subunit of Torpedo acetylcholine receptor. This peptide contains the sequence Arg-Arg-Ser-Ser which has been proposed as the site for phosphorylation of the acetylcholine receptor (AChR) by an endogenous cAMP-dependent protein kinase. We have shown that the synthetic peptide can be phosphorylated by the catalytic subunit of bovine heart cAMP-dependent protein kinase. Antibodies elicited against peptide 354-367 were shown to cross-react with native AChR and to bind specifically to the delta- and gamma-subunit as detected by immunoblotting. Furthermore, antipeptide antibodies were shown to inhibit specifically the cAMP-dependent phosphorylation of both the delta- and gamma-subunits. This suggests that the phosphorylation sites in the delta- and gamma-subunits are highly cross-reactive, and is in agreement with the demonstration that an endogenous cAMP-dependent kinase phosphorylates these two subunits, probably on homologous sequences. Tryptic digestion of the delta-subunit isolated from phosphorylated AChR yields a single 25-kd phosphorylated fragment. Immunoblotting experiments allowed us to map peptide 354-367 within this phosphorylated fragment.
Souroujon
M C
MC
Neumann
D
D
Pizzighella
S
S
Fridkin
M
M
Fuchs
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004557:Electric Organ; D007106:Immune Sera; D046911:Macromolecular Substances; D009842:Oligopeptides / Q000032:analysis; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D011950:Receptors, Cholinergic / Q000378:metabolism*; D014101:Torpedo; D014357:Trypsin
D007106:Immune Sera; D046911:Macromolecular Substances; D009842:Oligopeptides; D010446:Peptide Fragments; D011950:Receptors, Cholinergic; D011494:Protein Kinases; D014357:Trypsin