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 gamma and delta subunits. Two-dimensional phosphopeptide mapping of the phosphorylated gamma and delta 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 gamma subunit was phosphorylated at Ser-353, contained within the sequence Arg-Arg-Ser(P)-Ser-Phe-Ile and that the delta subunit was phosphorylated at Ser-361, contained within the sequence Arg-Ser-Ser(P)-Ser-Val-Gay-Tyr-Ser-Lys. Determination of the sites phosphorylated within the structure of the gamma and delta subunits should contribute to the molecular characterization of the regulation of desensitization of the nicotinic acetylcholine receptor by protein phosphorylation.
Yee
G H
GH
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021.
Huganir
R L
RL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D010449:Peptide Mapping; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D011494:Protein Kinases / Q000378:metabolism*; D011950:Receptors, Cholinergic / Q000378:metabolism*; D014101:Torpedo
D011950:Receptors, Cholinergic; D010768:Phosphoserine; D011494:Protein Kinases