Mapping of the cAMP-dependent phosphorylation sites on the acetylcholine receptor.

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Abstract
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.