Gastrin is a potent regulator of normal gastrointestinal cell growth and acid secretion; therefore elucidation of mechanisms that control its synthesis may be applied to understanding such disease states as neoplastic transformation and peptic ulceration. Although gastrin release from enriched antral G cell populations is known to be regulated by cholinergic agonists and the neuropeptide bombesin, the direct cellular mechanisms controlling gastrin synthesis at the level of transcription are poorly understood. This proposal addresses the hypothesis that direct activation of the Epidermal Growth Factor (EGF) receptor on antral G cells stimulates gastrin gene expression. The specific aims focus on EGF as a physiologic regulator of gastrin gene expression and use this observation to dissect the nuclear events that occur in response to growth factor stimulation. Enriched primary G cells will be isolated from dog and stimulated with EGF, Transforming Growth Factor alpha (TGFalpha), bombesin, and phorbol esters prior to measuring changes in gastrin mRNA on Northern blots. Nuclear runoff assays and intron probe analysis will be used to determine if the increase in gastrin mRNA levels is the result of an increase in new transcript synthesis or mRNA stability. To demonstrate that the antral G cell is capable of responding directly to EGF, the EGF receptor and gastrin antibodies were co-localized to the G cell in dog antral tissue. Prior work in a pituitary cell line (GH4) using gastrin-reporter gene constructs identified a novel DNA element, -68GGGGCGGGGTGGGGGG-53, that is required for both EGF and phorbol ester regulation of gastrin gene expression. Two nuclear proteins bind specifically to this element: Sp1 and a previously uncharacterized transcription factor called gERP (gastrin EGF Responsive Protein). The cDNA for Sp1 has been previously cloned. Therefore, the gERP cDNA will be cloned by screening an expression lambdagt11 phage library (Southwester method). To determine whether Sp1 and gERP bind separately or cooperatively to confer the EGF response point mutations of gERE will be tested in gel shift and transient transfection assays. Transcriptional control by both EGF and phorbol ester-activated protein kinase C (PKC) converge upon the gastrin EGF Response Element (gERE); therefore, it is likely that changes in the phosphorylation state of these regulatory proteins are responsible for activation of the gastrin gene. To determine if changes in the phosphorylation state of Sp1 or gERP alter binding to gERE, specific phosphatase inhibitors (e.g. okadaic acid) will be added to the gel shift assays. If blocking phosphatase activity does indeed alter transcription factor binding in vitro, then the effect of these phosphatase inhibitors on transient transformants containing gastrin-reporter constructs will be examined. Therefore these studies hope to explore the molecular mechanisms by which the gastrin is regulated, in particular by growth factors.