Determination of the tyrosine phosphorylation sites of the nicotinic acetylcholine receptor.
The peripheral nicotinic acetylcholine receptor (nAChR) is phosphorylated on tyrosine residues in vivo and in vitro at a high stoichiometry. We have previously reported that this tyrosine phosphorylation occurs on the beta, gamma, and delta subunits of the receptor and is implicated in both the modulation of the function of the receptor and localization of the receptor at the synapse. The specific tyrosine residue of each subunit which is phosphorylated is now identified. The endogenously phosphorylated nAChR from the electric organ of Torpedo californica was phosphorylated to maximal stoichiometry in vitro exclusively on tyrosine residues as indicated by phosphoamino acid analysis. Two-dimensional phosphopeptide maps of thermolysin limit digests of the isolated phosphorylated subunits indicated that each subunit is phosphorylated at a single site. To determine the site of tyrosine phosphorylation of the beta, gamma, and delta subunits, phosphorylated subunits were isolated and digested with trypsin. A single phosphotyrosine containing peptide from each subunit was purified by antiphosphotyrosine antibody affinity chromatography and reverse phase high performance liquid chromatography. The purified phosphopeptides were subjected to sequential Edman degradation and sequence analysis. Comparison of the phosphopeptide sequence data with the deduced amino acid sequence of each subunit indicated that Tyr-355 of beta, Tyr-364 of gamma, and Tyr-372 of delta are the sites of in vitro and in vivo tyrosine phosphorylation of the nAChR. Identification of these sites should facilitate further studies of the role of tyrosine phosphorylation in the regulation of receptor function.
Wagner
K
K
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Edson
K
K
Heginbotham
L
L
Post
M
M
Huganir
R L
RL
Czernik
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D004557:Electric Organ / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D007473:Ion Channels / Q000502:physiology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D019000:Phosphotyrosine; D011978:Receptors, Nicotinic / Q000302:isolation & purification / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D013570:Synaptic Membranes / Q000378:metabolism*; D014101:Torpedo; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis
D007473:Ion Channels; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010748:Phosphopeptides; D011978:Receptors, Nicotinic; D019000:Phosphotyrosine; D014443:Tyrosine