Wagner K, Edson K, Heginbotham L, Post M, Huganir R L, Czernik A J
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
J Biol Chem. 1991 Dec 15;266(35):23784-9.
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.
外周烟碱型乙酰胆碱受体（nAChR）在体内和体外均以高化学计量比在酪氨酸残基上发生磷酸化。我们之前报道过，这种酪氨酸磷酸化发生在受体的β、γ和δ亚基上，并且与受体功能的调节以及受体在突触处的定位都有关联。现在已确定每个亚基上发生磷酸化的特定酪氨酸残基。如磷酸氨基酸分析所示，来自加州电鳐电器官的内源性磷酸化nAChR在体外仅在酪氨酸残基上被磷酸化至最大化学计量比。对分离出的磷酸化亚基进行嗜热菌蛋白酶有限消化后的二维磷酸肽图谱表明，每个亚基在单个位点发生磷酸化。为了确定β、γ和δ亚基的酪氨酸磷酸化位点，分离出磷酸化亚基并用胰蛋白酶进行消化。通过抗磷酸酪氨酸抗体亲和色谱和反相高效液相色谱法从每个亚基中纯化出一个含磷酸酪氨酸的肽段。对纯化后的磷酸肽进行连续的埃德曼降解和序列分析。将磷酸肽序列数据与每个亚基的推导氨基酸序列进行比较，结果表明β亚基的Tyr-355、γ亚基的Tyr-364和δ亚基的Tyr-372是nAChR在体外和体内酪氨酸磷酸化的位点。这些位点的确定应有助于进一步研究酪氨酸磷酸化在受体功能调节中的作用。
