Phosphatidylinositol phosphatase activity of Phogrin regulates insulin secretion

描述(申请人提供)：胰岛素储存在胰腺β细胞的致密核心小泡(DCV)中。从囊泡释放胰岛素需要DCV膜与质膜的瞬时融合。这在一定程度上受DCV中的磷脂和跨膜蛋白的调节。Phogrin(IA-2β)是一种定位于分泌颗粒的跨膜蛋白酪氨酸磷酸酶样蛋白。这表明它在调节胰岛素分泌中起作用，但Phogrin的一个潜在的催化位点含有Asp，而不是预期的酪氨酸磷酸酶共识Ala，并且Phogrin的酶活性尚未见报道。我已获得的证据表明Phogrin能够对磷脂酰肌醇磷脂(PIP)进行脱磷，包括PI(3)P和PI(4，5)P2。结合PI(3)P和PI(4，5)P2的蛋白结构域可以将特定的蛋白质定位到正确的囊泡室，并改变结合蛋白的活性，从而调节分泌囊泡的形成、释放和再循环。我将测试这一假设，即Phogrin是一种PIPase，它通过调节分泌囊泡中的PIP水平和循环内含体来调节胰岛素的释放。我将在4个具体目标上检验这一假设： 目的1.体外测定全长Phogrin的PIPase活性。 目的2.检测Phogrin PIPase活性的药理抑制剂。 目的3.验证PKA对Phogrin的磷酸化作用可以使Phogrin在质膜失活，从而在胞吐和囊泡循环过程中不会使质膜和内体中的PIP发生水解性反应。 目的4.利用内源性Phogrin基因敲除来检验内源性Phogrin是一种 DCV PI(4，5)P2含量的决定因素，并在促分泌剂刺激的胞吐中发挥作用。 这些研究将为理解胰岛素分泌提供新的信息，并推动我们对糖尿病患者胰岛素释放分泌途径中哪些点可能被错误调节的认识。此外，我希望获得的数据将指导我未来的临床治疗研究，以纠正胰岛素分泌的异常步骤。

DESCRIPTION (provided by applicant): Insulin is stored in dense core vesicles (DCVs) in pancreatic beta cells. Release of insulin from a vesicle requires transient fusion of the DCV membrane with the plasma membrane. This is regulated, in part, by phospholipids and transmembrane proteins in the DCV. Phogrin (IA-2beta) is a transmembrane protein tyrosine phosphatase-like protein that is localized to secretory granules. This has suggested that it plays a role in regulating insulin secretion, but a potential catalyitc site of Phogrin contains Asp in place of an expected tyrosine phosphatase consensus Ala, and no enzymatic activity for Phogrin has ever been reported. I have obtained evidence that Phogrin is able to dephosphorylate phosphatidylinositol phospholipids (PIPs), including PI(3)P and PI(4,5)P2. Protein domains that bind PI(3)P and PI(4,5)P2 serve to localize specific proteins to the correct vesicle compartment and to alter the activity of the bound proteins and thus, function to regulate the formation, release, and recycling of secretory vesicles. I will test the hypothesis that Phogrin is a PIPase and that it plays a role in regulating insulin release by regulating PIP levels in secretory vesicles and recycling endosomes. I will test this hypothesis in 4 specific aims: Aim 1. Characterize the PIPase activity of full length Phogrin in vitro. Aim 2. Test pharmacologic inhibitors of the PIPase activity of Phogrin. Aim 3. Test the hypothesis that phosphorylation of Phogrin by PKA serves to inactivate Phogrin at the PM so that it does not hydrolyze PIPs in the PM and endosome during exocytosis and vesicle recycling. Aim 4. Use knockdown of endogenous Phogrin to test the hypothesis that endogenous Phogrin is a determinant of DCV PI(4,5)P2 content and plays a role in secretagogue-stimulated exocytosis. These studies will provide new information toward the understanding of insulin secretion and push forward our knowledge as to what points of the insulin release secretory pathway may be mis-regulated in diabetes. Further, I expect to obtain data which will guide my future studies toward clinical treatments to correct aberrant steps in insulin secretion.