Determination of the sites of cAMP-dependent phosphorylation on the nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor is a substrate for cAMP-dependent protein kinase both in vitro and in vivo. Recently, it has been demonstrated that phosphorylation of the nicotinic receptor by this kinase increases its rate of rapid desensitization. We now report the identification of the cAMP-dependent phosphorylation sites on the γ and δ subunits. Two-dimensional phosphopeptide mapping of the phosphorylated γ and δ subunits, after limit proteolysis with thermolysin, indicated that each subunit is phosphorylated on a single site. Phosphoamino acid analysis of the ³²P-labeled subunits demonstrates that phosphorylation had occurred exclusively on serine residues. Purified phosphorylated subunits were cleaved with cyanogen bromide and the resultant phosphopeptides were purified by reverse-phase high performance liquid chromatography. Shorter phosphopeptides, obtained by secondary digestion with trypsin, were purified and subjected to both automated gas-phase sequencing and manual Edman degradation. The results demonstrate that the γ subunit was phosphorylated at Ser-353, contained within the sequence Arg-Arg-Ser(P)-Ser-Phe-Ile and that the δ subunit was phosphorylated at Ser-361, contained within the sequence Arg-Ser-Ser(P)-Ser-Val-Gly-Tyr-Ile-Ser-Lys. Determination of the sites phosphorylated within the structure of the γ and δ subunits should contribute to the molecular characterization of the regulation of desensitization of the nicotinic acetylcholine receptor by protein phosphorylation.