The central hypothesis of our proposal is that molecular forms of peptide hormones have: 1) form-specific effects on target cells, 2) more than one function on the same or different target cells, 3) different intracellular routes of action mediating the different effects and 4) differences in the receptor characteristics on the various normal and malignant target cells. In the present grant request, the above hypotheses will be mainly tested by examining the cellular and intracellular actions of gastrin-releasing peptide/bombesin (GRP/BBS)-related peptides. BBS receptors (R) on normal (GI mucosal and pancreas) and malignant cells were observed to demonstrate species and target cell specific structural requirements for binding. The BBS-R on different target cells will be further characterized in terms of molecular size, binding characteristics, cellular localization, internalization of the hormone-R complexes and the receptor protein from different cells purified for the purposes of generating antibodies. These studies will help us to determine if similar or different types of BBS-R are present on normal and malignant target cells. Structure-specific effects of GRP-analogues, and a differential requirement of Ca++ for the release of SRIF versus gastrin was reported by us. The intracellular mechanism of action of BBS on the different normal target cells (along the GI tract and pancreas) and the malignant cells derived from these normal target cells will be investigated by determining the importance of: a) Ca++/phosphatidylinositol-mediated protein kinase C pathway, b) the adenylate cyclase-mediated protein kinase A pathway, c) the Ca++ calmodulin-dependent protein kinase pathway, and d) tyrosine kinase pathway. The relative importance of phosphorylation/dephosphorylation in the BBS-stimulated release of hormones will also be examined. These studies will help to delineate the differences, if any, in the intracellular routes of action of BBS/GRP-related peptides in the different target cells. The mechanisms of desensitization and resensitization of the target tissues to the action of various gut peptide hormones will be defined, and the regulation of specific binding sites for gastrin, CCK, BBS, and VIP, on the appropriate target cells will be investigated. A role of cholinergic, adrenergic and peptidergic pathways and other possible endocrine pathways on BBS-mediated release of CCK from duodenal cells and somatostatin from antral cells will be investigated, in vitro and in vivo models of study.
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