application) The mammalian regulatory peptide/neurotransmitter gastrin-releasing peptide (GRP) represents the specific, high affinity. ligand for the human gastrin-releasing peptide receptor (hGRP-R), a member of the mammalian bombesin receptor family. This family comprises three G protein-coupled receptors with the characteristic seven transmembrane protein structure. In addition to mediating important physiologic actions of GRP in the gastrointestinal tract, such as pancreatic enzyme secretion and gastric acid secretion, the hGRP-R also mediates signals of proliferation and differentiation in human intestinal and extraintestinal cells bot in vivo and in vitro. However, the underlying mechanisms and signaling factors responsible for cell proliferation through stimulation of the hGRP-R or other intracellular signaling molecules and their effect on hGRP-R gene expression remain poorly understood. Therefore, the studies in this proposal are directed at the elucidation of the molecular organization of the hGRP-R gene to ultimately identify mechanisms of interaction of signal molecules governing hGRP-R gene expression relevant to cellular proliferation in gastrointestinal malignancies. To accomplish this objective, this proposal will first elucidate the genomic organization of the hGRP-R in a genomic bacteriophage P1 clone to characterize the necessary structural basis for regulatory mechanisms of hGRP-R gene expression relevant to cellular growth. Secondly, a human gastrointestinal cell line that expresses detectable hGRP-R mRNA levels has been identified to reflect the native hGRP-R transcripts detected in the normal human pancreas, and it will be used for the complete characterization of hGRP-R transcripts. This cell line will also serve as a model system to characterize mechanisms of hGRP-R regulation by cAMP and phorbol esters, molecules that can activate signaling cascades of the protein kinase C (PKC) and mitogen activated protein kinase (MAPK) pathways. In the murine GRP-R model, these signaling cascades can be activated by high-affinity binding of the specific receptor ligand and are capable if initiating mitogenic signals in murine cells. In contrast, these pathways have not been examined for the human GRP-R with respect to their interaction on the gene regulatory level. The characterization of the hGRP-R gene will permit us to determine the minimal hGRP-R promoter sequence by cloning of hGRP-R- luciferase chimeric reporter plasmids and performing transient transfection assays in relevant gastrointestinal cell lines. Specific cis-regulatory elements in the core hGRP-R promoter will be examined for their ability to bind downstream molecules of the PKC and MAPK pathways and alter hGRP-R expression relevant to cellular proliferation. Collectively, the studies will provide invaluable information regarding the molecular organization of the hGRP-R gene and will determine regulatory mechanisms of its expression. Due to its important relationship to mitogenic and other factors involved in carcinogenesis, these studies will greatly contribute to the general understanding of malignant transformation in gastrointestinal and other tissues.
