Phosphatidylinositol phosphatase activity of Phogrin regulates insulin secretion

描述（由申请人提供）：胰岛素储存在胰腺β细胞的致密核心囊泡（DCVs）中。胰岛素从囊泡中释放需要DCV膜与质膜的短暂融合。这部分是由磷脂和跨膜蛋白在DCV中调节的。Phogrin （ia -2 β）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的潜在催化位点含有Asp，而不是预期的酪氨酸磷酸酶Ala，并且Phogrin的酶活性尚未报道。我已经获得了Phogrin能够使磷脂酰肌醇磷脂（PIPs）去磷酸化的证据，包括PI(3)P和PI(4,5)P2。结合PI(3)P和PI(4,5)P2的蛋白结构域将特定蛋白定位到正确的囊泡区，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。我将验证Phogrin是一种PIPase的假设，它通过调节分泌囊泡中的PIP水平和再循环内体来调节胰岛素释放。我将用4个具体目标来检验这一假设：

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.