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

我们试图鉴定β细胞素受体的克隆。1. 我们画了两列。其中一种含有全长Betacellulin （BTC/1-80）。另一种含有β细胞素（BTG2-76），其含量足以引起胰腺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似乎结合并使用EGF受体和EGF。我们没有发现任何其他含有受体结构的候选分子。2. 我们推测BTC和EGF刺激可能存在磷酸化分子的差异。我们用BTC或EGF刺激βHC9细胞，酪氨酸磷酸化分子通过磷酸化酪氨酸抗体的免疫沉淀设计被拉下。对SDS-PAGE上BTC刺激特有的分离带进行分离并进行LC/MS分析。最后我们获得了一个含有磷脂酶D活性位点基序的分子，该基序具有EGFR磷酸化位点。我们认为该分子通过PLD2/SostRas的Ras激活途径调控细胞增殖。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.