Gastrointestinal (GI) peptides, including mammalian bombesin-like peptides, integrate secretory, contractile and proliferative functions in the GI tract. The broad, long-term objectives of this proposal is to elucidate the signal transduction pathways that mediate GI peptide-induced cell proliferation. GI peptides exert their characteristic effects on cellular processes by binding to specific G protein-coupled receptors (GPCR). In addition to signal via classic second messengers (e.g., Ca2+, DAG, cAMP) and the consequent stimulation of serine/threonine protein kinase cascades, an emerging theme is that GPCR agonists also induce tyrosine phosphorylation of multiple proteins. We identified the non-receptor tyrosine kinase p125 focal adhesion kinase (FAK) and the adaptor proteins p130 targets of GPCR-stimulated tyrosine phosphorylation. Bombesin stimulation also induces raid Src activation and epidermal growth factor receptor (EGFR) transactivation. Our central hypothesis is that tyrosine phosphorylation plays a critical role in GPCR-induced cellular migration and mitogenesis. An additional hypothesis is that these tyrosine phosphorylation cascades are operational in intestinal epithelial cells. This proposal will pursue the following specific hypotheses and aims: Hypothesis 1. GI peptide induce tyrosine phosphorylation of focal adhesion proteins and assembly of signaling complexes via a cytoskeleton dependent pathways.
Aims emanating from hypothesis 1: 1a) Characterize FAK phosphorylation sites and the formation of FAK signaling complexes using the endogenously pressed bombesin/GRP receptor as a model for gastrointestinal GPCRs. 1b) Determine the upstream components in the pathway(s) leading to focal adhesion protein tyrosine phosphorylation and to the assembly of FAK signaling complexes. 1c) Characterize the tyrosine phosphorylation of focal adhesion proteins and the assembly of signaling complexes in the intestinal epithelial cell line IEC-6, a well established model of restitution. 1d) Characterize signal transduction and mitogenesis in cells expressing wild type or dominant-negative forms of FAK. Hypothesis 2: EGFR transactivation mediates a novel growth-promoting signal transduction pathway in the action of GI peptides.
Aims emanating from hypothesis 2: 2a) Id4entify the intracellular mechanisms leading to rapid GI peptide-induced EGFR transactivation. 2b) Determine the role of EGFR transactivation in signaling GI peptide-mediated mitogenesis. 2c) Characterize GPCR-induced transactivation of EGFR in IEC-6 cells.
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