Exocytosis involves the delivery of secretory products packaged in membrane-bound vesicles to the extracellular space. Exocytosis has been recognized to be stimulable, called regulated exocytosis, and occurs constitutively without stimulation universally in all cells. In the last few years, several proteins associated with secretory vesicles have been identified and some characterized. However, the actual biochemical mechanism of vesicle targeting, docking and fusion with the plasma membrane is unclear. Over the last decade, a number of studies have implicated guanine nucleotide binding proteins (GTP-binding proteins) in intracellular vesicle traffic. Until two years ago, the only GTP-binding proteins implicated in intracellular vesicle traffic were the monomeric, low molecular weight GTP-binding proteins (Sec, ypt, rab, arf, Sar, and Sas family of proteins) which belong to the ras-superfamily. Recent studies however suggests the involvement of heterotrimeric GTP-binding proteins (G-proteins) in intracellular vesicle traffic. Recent studies also suggest the regulative interaction between monomeric and G-proteins in vesicle transport process. Over the last two years, our studies have identified the association of both monomeric as well as heterotrimeric G- proteins with zymogen granule membranes (ZGM) from rat exocrine pancreas. Our studies demonstrates the involvement of one of the rab's (rab3) in regulated exocytosis from exocrine pancreas. Our recent studies have also identified a novel Sec4-like protein (MSec4), which may be involved in constitutive exocytosis from mammalian cells.
The specific aim of this study is to understand the role of G-proteins in exocytosis from the exocrine pancreas, with the long term objective of understanding the biochemical mechanism of exocytosis in the tissue. In view of this, the molecular mechanism of interaction between G-proteins and its interactive proteins regulating exocytosis of zymogen granules from exocrine pancreas will be investigated. Studies will be carried out to further identify and characterize heterotrimeric G-proteins associated with exocrine pancreatic ZGM's. Azido-GTP binding followed by gel electrophoresis and immunoblot analysis using antibodies to specific GTP-binding proteins will be performed. In vitro and in vivo functional studies will be carried out both in the presence or absence of molecules regulating specific ZGM associated G-protein, which will involve the introduction of specific peptides, salts, toxins and antibodies into Streptolysin O permeabilized acini, or into cell-free ZG and plasma membrane fusion assays. Additionally, chemical cross-linking studies followed by immunoprecipitation using antibodies to specific monomeric and G-proteins will be carried out. Such studies will enable us to define the proximity and interaction between the two classes of guanine nucleotide binding proteins as well as other regulatory proteins. Understanding the role of the secretory granule membrane-associated G-proteins in exocytosis will help us understand better this fundamental cellular process which when impaired leads to a number of diseases.
