Phosphatidylinositol phosphatase activity of Phogrin regulates insulin secretion

Phogrin 的磷脂酰肌醇磷酸酶活性调节胰岛素分泌

• 批准号: 7697937
• 负责人: LESLIE ANN CAROMILE
• 金额: $ 1.36万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2009-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7697937
• 关键词: Binding Proteins; Cell membrane; Clinical Treatment; Consensus; Cyclic AMP-Dependent Protein Kinases; Data; Dense Core Vesicle; Diabetes Mellitus; Endosomes; Exocytosis; Future; Hydrolysis; In Vitro; Insulin; Integral Membrane Protein; Knowledge; Length; Membrane; PTPRN gene; Pathway interactions; Phosphatidylinositols; Phospholipids; Phosphoric Monoester Hydrolases; Phosphorylation; Play; Protein Binding Domain; Protein Tyrosine Phosphatase; Proteins; Recycling; Reporting; Role; Secretory Vesicles; Site; Structure of beta Cell of islet; Testing; Vesicle; inhibitor/antagonist; insulin secretion

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.

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（DCV）中。从囊泡中释放胰岛素需要 DCV 膜与质膜的短暂融合。这部分是由 DCV 中的磷脂和跨膜蛋白调节的。 Phogrin (IA-2beta) 是一种跨膜蛋白酪氨酸磷酸酶样蛋白，定位于分泌颗粒。这表明它在调节胰岛素分泌中发挥作用，但 Phogrin 的潜在催化位点含有天冬氨酸，取代了预期的酪氨酸磷酸酶共有 Ala，并且尚未报道过 Phogrin 的酶活性。我已获得证据表明 Phogrin 能够使磷脂酰肌醇磷脂 (PIP) 去磷酸化，包括 PI(3)P 和 PI(4,5)P2。结合 PI(3)P 和 PI(4,5)P2 的蛋白质结构域用于将特定蛋白质定位到正确的囊泡区室并改变结合蛋白质的活性，从而发挥调节分泌囊泡。我将检验这样的假设：Phogrin 是一种 PIPase，它通过调节分泌囊泡中的 PIP 水平和回收内体来调节胰岛素释放。我将通过 4 个具体目标来检验这个假设： 目标 1. 表征全长 Phogrin 的体外 PIPase 活性。 目标 2. 测试 Phogrin 的 PIPase 活性的药理学抑制剂。 目标 3. 测试以下假设：PKA 对 Phogrin 进行磷酸化，从而使 PM 上的 Phogrin 失活，使其在胞吐作用和囊泡回收过程中不会水解 PM 和内体中的 PIP。 目标 4. 使用内源性 Phogrin 的敲低来检验内源性 Phogrin 是一种假设 DCV PI(4,5)P2 含量的决定因素，并在促分泌剂刺激的胞吐作用中发挥作用。 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中胰岛素释放分泌途径的哪些点可能被错误调节的认识。此外，我希望获得数据来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。