Determination of the tyrosine phosphorylation sites of the nicotinic acetylcholine receptor

Kathryn Rae Wagner, Kathryn Edson, Lise Heginbotham, Marc Post, Richard L Huganir, Andrew J. Czernik

Research output: Contribution to journalArticle

Abstract

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 β, γ, and γ 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 β, γ, and δ 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 β, Tyr-364 of γ, and Tyr-372 of δ 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.

Original languageEnglish (US)
Pages (from-to)23784-23789
Number of pages6
JournalJournal of Biological Chemistry
Volume266
Issue number35
StatePublished - Dec 15 1991

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Phosphorylation
Nicotinic Receptors
Tyrosine
Phosphopeptides
Stoichiometry
Phosphoamino Acids
Thermolysin
Electric Organ
Affinity chromatography
Torpedo
Phosphotyrosine
Antibody Affinity
High performance liquid chromatography
Reverse-Phase Chromatography
Affinity Chromatography
Synapses
Trypsin
Sequence Analysis
Amino Acid Sequence
High Pressure Liquid Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Determination of the tyrosine phosphorylation sites of the nicotinic acetylcholine receptor. / Wagner, Kathryn Rae; Edson, Kathryn; Heginbotham, Lise; Post, Marc; Huganir, Richard L; Czernik, Andrew J.

In: Journal of Biological Chemistry, Vol. 266, No. 35, 15.12.1991, p. 23784-23789.

Research output: Contribution to journalArticle

Wagner, Kathryn Rae ; Edson, Kathryn ; Heginbotham, Lise ; Post, Marc ; Huganir, Richard L ; Czernik, Andrew J. / Determination of the tyrosine phosphorylation sites of the nicotinic acetylcholine receptor. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 35. pp. 23784-23789.
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