Studies on the autophosphorylation of the insulin receptor from human placenta. Analysis of the sites phosphorylated by two-dimensional peptide mapping.
Studies on the autophosphorylation of the insulin receptor from human placenta. Analysis of the sites phosphorylated by two-dimensional peptide mapping.
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人胎盘胰岛素受体自身磷酸化的研究。
DOI：
10.1042/bj2520607
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发表时间：
1988
期刊：
影响因子：
--
通讯作者：
R. Denton
中科院分区：
文献类型：
--
作者：
J. Tavaré;R. Denton
1. 部分纯化的人胎盘胰岛素受体制剂与[γ - 32p]ATP在存在或不存在胰岛素的情况下孵育。然后分离32P标记的胰岛素受体β亚基，用胰蛋白酶和蛋白酶V8进行裂解，并通过薄层电泳和层析分析生成的[32P]磷酸肽。该方法表明，胰岛素刺激胰岛素受体β亚基在体外至少七个酪氨酸残基上的自磷酸化，分布在三个不同的结构域。2. 其中一个结构域（结构域2）包含酪氨酸残基1146、1150和1151，其磷酸化速度最快，可被胰蛋白酶在Arg-1143和Lys-1153或Lys-1156位点切割为单、二和三磷酸化肽。该结构域的多次磷酸化似乎部分抑制了胰蛋白酶对Lys-1153的切割。3. 在第二个结构域（结构域3）中，在1316和1322位置上含有两个磷酸化的酪氨酸残基，酪氨酸的磷酸化速度比结构域2中的酪氨酸磷酸化速度慢。该结构域靠近β -亚基多肽链的c端。4. 在结构域2和3之后，至少还有两个酪氨酸残基被磷酸化。这些残基可能在含有酪氨酸953、960和972的质膜内表面附近的区域内残留，但仍需要确凿的证据。5. 本研究中使用的二维薄层分析来研究胰岛素受体磷酸化，与以前基于反相色谱的方法相比，具有几个优点。它允许更高分辨率的32p标记的色氨酸，当与放射自显影相结合时，相当敏感。该方法可以很容易地适应于研究完整细胞内胰岛素受体的磷酸化。
1. A partially purified preparation of human placental insulin receptors was incubated with [gamma-32P]ATP in the presence or absence of insulin. The 32P-labelled insulin-receptor beta-subunits were then isolated, cleaved with trypsin followed by protease V8 and the [32P]phosphopeptides generated were analysed by thin layer electrophoresis and chromatography. This approach revealed that insulin stimulates autophosphorylation of the insulin-receptor beta-subunit in vitro on at least seven tyrosine residues distributed among three distinct domains. 2. One domain (domain 2), containing tyrosine residues 1146, 1150 and 1151 was the most rapidly phosphorylated and could be recovered as mono-, di- and triphosphorylated peptides cleaved by trypsin at Arg-1143 and either Lys-1153 or Lys-1156. Multiple phosphorylation of this domain appears to partially inhibit the cleavage at Lys-1153 by trypsin. 3. In a second domain (domain 3) containing two phosphorylated tyrosine residues at positions 1316 and 1322 the tyrosines were phosphorylated more slowly than those in domain 2. This domain is close to the C-terminus of the beta-subunit polypeptide chain. 4. At least two further tyrosine residues appeared to be phosphorylated after those in domains 2 and 3. These residues probably residue within a domain lying in close proximity to the inner face of the plasma membrane containing tyrosines 953, 960 and 972, but conclusive evidence is still required. 5. The two-dimensional thin-layer analysis employed in this study to investigate insulin-receptor phosphorylation has several advantages over previous methods based on reverse-phase chromatography. It allows greater resolution of 32P-labelled tryptic peptides and, when coupled to radioautography, is considerably more sensitive. The approach can be readily adapted to study phosphorylation of the insulin receptor within intact cells.