The regulated secretion of polypeptide hormones and peptide neurotransmitters occurs though the fusion of pre-existing dense cored vesicles (termed DCVs or granules) with the plasma membrane and exocytosis of their contents. Integral membrane proteins of these organelles are thought to play a central role in the morphogenesis, maturation and senescence of these organelles and their interactions with cytoskeleton and plasma membrane during exocytosis and subsequent retrieval of membrane for incorporation into new granules. The identification of such molecules has however been impeded by their low abundance in mammalian tissues and lack of tractable Genetic models in lower organisms. We have identified an insulin granule membrane protein tyrosine phosphatase (P.T..P.) family member termed phogrin which is broadly distributed in neuroendocrine tissues and localized specifically to dense core vesicles. Preliminary data show it can be used as a reporter molecule for granule movement and membrane trafficking and to monitor the immediate cytosolic ionic environment of the granule. Phogrin undergoes rapid reversible Ca2+ -dependent phosphorylation on Ser/Thr residues in response to secretory stimuli in intact pancreatic Beta-cells. We hypothesize that phogrin is a key element in a signal transduction pathway that relays information from the umen of the DCV to the cytoplasm. We furthermore postulate that covalent modification of the Ser-rich region of the cytosolic domain of the protein in response to secretagogues initiates the interaction of phogrin with other molecules of the secretory machinery which directly or indirectly interact with cytoskeletal motor proteins and so regulate the recruitment of granules to the site of exocytosis, In the proposed study we aim to: 1) Define the functional elements of the phogrin molecule in the context of the sorting of the protein to the regulated secretory pathway and regulation of the movement of granules within the cytoplasm in response to stimuli and secretion. 2) Map the sites of phosphorylation in the molecule, define which sites are critical in the response to secretagogues and identify the enzymes linking changes in intracellular Ca2+ to phosphorylation of theses residues. 3)Evaluate the functional importance of phogrin in the secretory pathway and determine the role of phosphorylation of phogrin in the regulation of DCV movement, exocytosis and recycling 4) Gain insight into the molecular mechanisms by which phogrin signals to the cytoplasm and interacts with the cytoskeleton. These investigations have direct bearing on understanding the mechanism of stimulus secretion coupling of insulin release and hence the pathogenesis of type 2 diabetes. Phogrin is of additional interest in type 1 diabetes as it is a major autoantigen.
