SECRETORY GRANULE MEMBRANE IN REGULATED EXOCYTOSIS

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This proposal seeks, over the next five years, to define the role of the secretory granule membrane in the regulation of exocrine secretion. The granule membrane compartment contains a unique set of compositionally related proteins common to different exocrine tissues. It is likely that some of these proteins are functionally involved in regulating secretion. Indeed, the identification by several investigators of neurohumorally- regulated secretory granule membrane phosphoproteins supports this hypothesis. Further, recent evidence shows a close correlation between the phosphorylation of specific granule membrane proteins and enzyme secretion. This project is designed to characterize, in the rate model, the role of secretory granule membrane phosphoproteins in the regulation of exocrine secretion. Information obtained from this project may contribute to our understanding of clinically-related disorders of exocytosis such as cystic fibrosis and experimental pancreatitis. There are four specific aims and methodological approaches to this proposal. First, the relationship between the phosphorylation of specific granule membrane proteins and secretion in intact cells will be examined using an antiserum raised against exocrine secretory granule membrane proteins. A close correlation between regulated protein phosphorylation and enzyme secretion will serve to identify physiologically important phosphoproteins for subsequent analysis. Second, a rapid granule purification procedure will be used to confirm localization of these phosphoproteins to one of the secretory compartments. Third, the phosphoproteins will be structurally characterized to determine their degree of conservation among secretory systems. Fourth, in vitro phosphorylation techniques will be employed to assign given second messenger pathways to specific phosphorylation events. Lastly, monospecific polyclonal antibodies will be raised to specific phosphoproteins for use in determining the precise subcellular localization of these species as well as to prepare affinity columns for the purification of large enough quantities of protein for amino acid sequencing.