Development of a new therapy for diabetes mellitus through Betacellulin receptor cloning

我们试图鉴定β-细胞蛋白受体的克隆。1.我们做了两列。其中一个含有全长的Betacellin(BTC/1-80)。另一种含有贝塔细胞蛋白(BTG2-76)，其含量足以诱导胰岛β细胞分化。我们重点研究了不与BTC-1-80柱结合但与BTC/24-76柱结合的分子。我们可以获得EGF受体，BTC似乎结合和利用了EGF受体和EGF。我们找不到任何其他含有受体结构的候选分子。2.我们推测BTC和EGF刺激的磷酸化分子可能存在差异。我们用牛血清白蛋白或表皮生长因子刺激β细胞，并用磷酸化酪氨酸抗体设计免疫沉淀法，将酪氨酸磷酸化的分子拉下。提取BTC刺激所特有的SDS-PAGE条带，进行LC/MS分析。最后，我们获得了一个含有带有EGFR磷酸化位点的磷脂酶D活性中心基序的分子。我们认为该分子通过PLD2/SostRas的Ras激活途径调节细胞增殖。下一步计划进行进一步调查。

We tried to identify the Betacellulin receptor cloning. 1. We made two columns. The one contains Betacellulin (BTC/1-80) which is full length. The other one contains Betacellulin (BTG2-76) which is enough portion to evoke pancreatic beta cell differentiation. We focused on the molecule which not binds to BTC-1-80 column but binds to BTC/24-76 column. We could obtain EGF receptor and BTC seems to bind and use EGF receptor as well as EGF. We could not find any other candidate molecule containing receptor structure. 2. We speculated that there might be difference of phosphorylated molecule between BTC and EGF stimuli. We stimulated βHC9 cells with either BTC or EGF and tyrosine-phosphorylated molecules were pulled down by the design of immunoprecipitation with phosphotyrosine antibody. The separated band on SDS-PAGE, which is just unique for BTC stimulation, was isolated and processed LC/MS analysis. Finally we obtained a molecule containing phospholipase D active site motif with EGFR phosphorylation site. We considered that this molecule is regulating cell proliferation through Ras activation pathway of PLD2/SostRas. Further investigation is planned as a next step.