Bombesin (BBS) and its mammalian counterpart, gastrin-releasing peptide (GRP) are important peptides that affect numerous aspects of intestinal function that include: 1) stimulation of peptide release, 2) increase in gene expression, and 3) stimulation of normal and neoplastic gut growth. Receptors that bind BBS are members of the G protein receptor superfamily and include the GRP-receptor (GRP-R) and the neuromedin-B receptor (NMB- R). Key questions remain that are crucial for understanding the biologic effects of BBS, in particular, and all GI hormones, in general. They include, what are the mechanisms that regulate the diverse cellular functions produced by a peptide when it binds to its receptor and are identical or separate second messenger pathways responsible for peptide release, alterations in gene expression and cell growth? The major focus of this project is to define the intracellular mechanisms that regulate the multiple biologic effects of BBS in the gut. To accomplish this goal we have established a unique cell model by transfection of functional BBS receptors into our previously-characterized human endocrine cell line, BON, which produces the hormones neurotensin (NT), chromogranin A (CGA) and pancreastatin. Our preliminary findings demonstrate that BBS increases peptide release, alters NT gene expression and stimulates cell growth of these BON transfectants. In addition, we show that BBS stimulates the growth of the human gastric cancer cell line SIIA through a receptor other than GRP-R or NMB-R. Furthermore, our in vivo studies demonstrate that BBS affects normal and neoplastic gut tissues in a differential and cell-specific fashion. Taken together, these results indicate that BBS acts through multiple cell-specific signal transduction pathways to regulate cellular function. Based upon our findings, the central hypothesis of this proposal is that GI hormones regulate peptide release, gene expression and growth through specific receptors and post- receptor mechanisms. To examine this hypothesis we have planned experiments with the following Specific Aims: 1) We will characterize signal transduction pathways through which BBS regulates hormone secretion. To accomplish this goal, we will use our transfected BON cell clones to further delineate, in a systematic fashion, the specific signal transduction pathways responsible for BBS-mediated hormone release. 2) We will determine the cellular factors important for BBS-mediated changes in peptide gene expression. To accomplish this goal, we will identify the mechanisms responsible for BBS-mediated increases in NT gene expression. 3) We will examine the receptors and intracellular mechanisms responsible for the trophic effects of BBS. To accomplish this goal, we will assess the effects of BBS on our cell-established models of BBS-mediated cellular proliferation. The long-term goal of this proposal is to systematically define the intracellular mechanisms that regulate the cellular effects of BBS utilizing novel cell model systems that have been established in our laboratory. An in-depth identification of the signal transduction systems and molecular mechanisms that are responsible for these multiple cellular functions are crucial to our overall understanding of the diverse biologic effects exerted by the GI hormones.
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