This revised application seeks to understand the interactions between pancreatic acinar cells and receptors for bombesin (Bn) and cholecystokinin (CCKA) or gastrin (CCKB). Pancreatic acinar cells are critical for digestion of food and are involved in pathological states including pancreatitis and pancreatic cancer. Bn and CCK receptors regulate acinar cell function and are themselves regulated by acinar cell mechanisms. The molecular mechanisms and the structural features of Bn and CCK receptors that influence acinar cell function have not been identified. The first specific aim of this proposal is to determine the molecular basis for differences in Bn, CCKA, and CCKB coupling to pancreatic acinar biological functions and cell signaling. The functioning of Bn, CCKA, and CCKB receptors from humans and rodents will be compared in acinar cells. For this purpose, gene transfer techniques, wither adenoviral-based vectors or transgenic animals, will be employed. The effects of expressing those receptors in acinar cells on receptor coupling to biological responses, including secretion, protein synthesis, and the generation of inflammatory mediators, and the signaling mechanisms associated with each of those responses will be examined. These studies will allow the first direct comparison between different receptor subtypes and receptors from different species in an acinar cell environment. Mutant receptors will be utilized to determine the structural basis of receptor coupling. The second specific aim is to determine the cellular mechanisms and receptor structural features involved in receptor trafficking in pancreatic acinar cells. Nothing is known concerning CCKB or Bn receptor trafficking in pancreatic acinar cells. CCKA receptors are immobilized on the acinar cell surface by agonist occupation but are not internalized. This pattern is unique to the acinar cell as these receptors are internalized in fibroblast models. The trafficking characteristics of human and rodent CKB and Bn receptors will be examined in pancreatic acinar cells employing biochemical and imaging techniques. Then, the role of specific domains and sites in receptor trafficking will be examined. Also, the subset of molecules important in receptor trafficking will be explored, including G protein-coupled receptor kinases (GRKs) and arrestins, in pancreatic acinar cells from humans and mice. The effects on receptor trafficking of over-expressing the relevant isoforms of these molecules and their dominant negative counterparts, or anti-sense constructs will be examined. The third specific aim will determine the cellular mechanisms and receptor structural features involved in receptor desensitization in pancreatic acinar cells. Little is known about the desensitization characteristics or molecular mechanisms involved in the desensitization of Bn or CCK receptors. The desensitization characteristics of the different receptors will be defined on pancreatic acinar cell biological responses and cell signals. Mutant receptors will be employed to determine the structural basis for receptor desensitization. Also, the effects of manipulating GRKs and arrestins on desensitization will be examined on acinar cell biological responses and associated intracellular signals. Together these studies will provide important insights into the mechanisms regulating pancreatic acinar cell function.
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