Biosynthesis and Processing of Peptide Hormone

DESCRIPTION (provided by applicant): The selective packaging of peptide hormone precursors into secretory vesicles is essential for hormone biosynthesis. In the trans Golgi network (TGN), the precursors are sorted into nascent secretory granules where they are processed to generate bioactive polypeptides. Data from our laboratory has demonstrated that the maintenance of Golgi structure and release of hormone containing vesicles from the TGN requires phosphatidic acid (PA), the product of phospholipase D (PLD)-mediated phosphatidylcholine hydrolysis, as well as synthesis of the inositol phospholipids phosphatidylinositol (4) phosphate, PI(4)P, and phosphatidylinositol (4,5) bisphosphate, PI(4,5)P2. The goal of this proposal is to understand the mechanisms whereby these lipids regulate Golgi structure, budding of secretory vesicles from the TGN and hormone processing. Aim 1: The role of PI(4)P and PI(4,5)P2 in maintaining Golgi structure and function in endocrine cells: We will test the hypothesis that in pituitary GH3 cells both PI(4)P and PI(4,5)P2 are required to maintain Golgi architecture whereas Pi(4,5)P2 also mediates release of post-Golgi vesicles and trafficking to the plasma membrane. Aim I1: The Regulation of Golgi PI(4,5)P2 synthesis: Surprisingly, the Golgi apparatus appears to possess little PI(4,5)P2 and its level maybe tightly controlled, in part, by a potent Pl(4,5)P2-5-phosphatase activity that we have identified. We will test the hypothesis that PI(4,5)P2 synthesis is regulated locally by recruitment of specific PI(4)P 5-kinases to the Golgi apparatus and by the activity of the Pl(4,5)P2-5-phosphatase. Aim III. To determine the role of PLD isoforms and PA in maintaining Golgi structure: PA has been implicated in vesicle trafficking, signal transduction and regulation of PI(4,5)P2 synthesis. We demonstrated in endocrine cells that PLD1 and -2, which generate PA, localize to different regions of the Golgi apparatus. We will determine the mechanism of PLD recruitment to the Golgi apparatus and use novel PA reporter proteins to demonstrate PA itself localizes to the Golgi apparatus. Although multiple enzymes involved in phospholipid biosynthesis have been characterized, in endocrine cells relatively little is known whereby the different activities are coordinated and integrated in the Golgi apparatus to control the formation of nascent secretory vesicles. Consequently, understanding the mechanism of secretory vesicle budding will facilitate the rational design of drugs that could be used to enhance or diminish hormone secretion in a variety of pathological conditions including diabetes.