Molecular Mechanisms of Proglucagon Sorting to the Regulated Secretory Pathway

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Background: Peptide hormones are synthesized in the endoplasmic reticulum as larger precursors or prohormones, transported through the Golgi, and sorted to the secretory granules of the regulated secretory pathway. Within these granules, prohormones are processed to their constituent peptide hormones and stored until a stimulus triggers their release. Proglucagon is the precursor of the pancreatic hormone glucagon and the intestinal hormones, glucagon-like peptide (GLP)-1 and -2. These peptides must be stored in secretory granules in order to be secreted in response to nutrients. Glucagon, the major glucose counter-regulatory hormone, is secreted in response to low blood glucose levels in order to maintain euglycemia. Both GLP-1 and GLP-2 are secreted in response to nutrient ingestion; GLP-1 stimulates glucose-dependent insulin secretion, and GLP-2 increases intestinal blood flow and nutrient absorption. My research program focuses on identifying the molecular mechanisms by which proglucagon is directed to secretory granules.***Progress: We have shown that proglucagon may interact with a sorting receptor, CPE, to be targeted to granules in pancreatic alpha cells, and that another receptor may be required in intestinal L cells. We have also identified specific sorting signals within the structures of glucagon and GLP-1 that direct proglucagon into granules. We will continue to characterize the molecular mechanisms that govern the sorting of proglucagon by using proteomics and quantitative super-resolution microscopy.***Specific Aims:***1. To characterize the lead candidates as sorting receptors for proglucagon in alpha cells. Our work indicates that CPE may be a sorting receptor in alpha cells but not L cells. We have additional data suggesting that the GLP-1 sorting signal can bind to CgA. We will determine if CPE and/or CgA directly interact with proglucagon. ***2. To identify clusters of proteins that interact with proglucagon sorting signals. We will use affinity purification-mass spectrometry to identify novel players in the sorting of proglucagon to granules in alpha and L cells. We will validate our findings using super-resolution microscopy and siRNA loss-of-function experiments.***3. To identify protein networks within secretory granules that regulate proglucagon sorting. Proteomic analysis of pancreatic alpha cell secretory granules will reveal novel networks involved in the post-translational processing and sorting of proglucagon, and will yield information on granule composition, architecture and biogenesis. ***Significance: Glucagon, GLP-1 and GLP-2 are key regulators of nutrient homeostasis. Correct sorting of these peptides to secretory granules is required for the alpha and L cells' response to nutrients. We will be using state-of-the-art quantitative techniques to investigate the molecular mechanisms that govern the intracellular trafficking of proglucagon. *****