The overall aim of this research is to understand how regulatory neurotransmitters and gastrointestinal hormones act through changes in intracellular free Ca2+ to bring about digestive enzyme secretion by pancreatic acinar cells. This proposal focuses on the mechanisms involved in the terminal steps in secretion culminating in exocytosis. Using a proteomics discovery strategy, we have identified a number of small GTP binding proteins on the zymogen granule including RabSD, Rab6, Rab11, Rab27B and Rap1. Since Rabs are believed to regulate vesicular trafficking by organizing and regulating effector proteins the focus of this work includes two of these Rab molecules, RabSD and Rab27B as well as Rap1.Two other small G proteins, Rho and Rac which regulate the actin cytoskeleton will also be studied. The overall goal is to determine the role of each of these G proteins in the series of sequential processes by which zymogen granules are brought to the apical membrane, become fusion competent and undergo exocytosis.
Specific aims of this proposal include: 1) What is the extent of activation (GTPliganded form) of Rab27B andRap1 on zymogen granules and is it increased by secretagogues such as cholecystokinin (CCK) and acetylcholine? Is Rap1important for secretion as has been shown for RabSD and 27B? Does Rap1 activation mediate the secretory stimulation by cyclic AMP? 2) What are the GEFs or other regulatory protein involved in activating the three granule small G proteins, Rho and Rac? Are they activated by specific heterotrimeric G proteins and/or by intracellular messengers such as Ca2+? Is a cyclic AMP activated GEF (Epac) involved in activating Rap1? 3) What are the downstream effector proteins for RabSD, Rab27B and Rap1? Are linker proteins of the Sip or Slac families involved? Is there a relation of specific small G proteins to myosin Vc? to Noc2? Which small G proteins directly or indirectly regulate SNARE complexes? This work will lead to better molecular understanding of the regulation of digestive enzyme secretion in acinar cells and potential sites for the pathological regulation that occurs in experimental pancreatitis. Relevance to public health: the pancreas is the major organ that secretes digestive breakdown of food and assimilation of nutrients. If these enzymes are not inflammatory disease pancreatitis can result. The present work is directed at understanding of the proteins involved in normal digestive enzyme secretion.
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