GI PEPTIDE SIGNALING THROUGH TYROSINE PHOSPHORYLATION

胃肠道（GI）肽，包括哺乳动物炸弹蛋白样肽，在胃肠道中具有分泌、收缩和增殖功能。本研究的长远目标是阐明介导GI肽诱导的细胞增殖的信号转导途径。GI肽通过与特定的G蛋白偶联受体（GPCR）结合，在细胞过程中发挥其特有的作用。除了通过经典的第二信使（例如，Ca2+， DAG, cAMP）和随之而来的丝氨酸/苏氨酸蛋白激酶级联刺激信号外，一个新兴的主题是GPCR激动剂还诱导多种蛋白质的酪氨酸磷酸化。我们确定了非受体酪氨酸激酶p125黏附激酶（FAK）和衔接蛋白p130是gpcr刺激酪氨酸磷酸化的靶点。Bombesin刺激还可诱导raidsrc激活和表皮生长因子受体（EGFR）转激活。我们的中心假设是酪氨酸磷酸化在gpcr诱导的细胞迁移和有丝分裂发生中起关键作用。另一种假设是这些酪氨酸磷酸化级联反应在肠上皮细胞中起作用。本提案将追求以下具体假设和目标：假设1。GI肽通过细胞骨架依赖途径诱导黏附蛋白的酪氨酸磷酸化和信号复合物的组装。假设1:1a)利用内源性加压的bombesin/GRP受体作为胃肠道gpcr模型，表征FAK磷酸化位点和FAK信号复合物的形成。1b)确定导致局灶黏附蛋白酪氨酸磷酸化和FAK信号复合物组装的途径中的上游组分。1c)描述肠上皮细胞系IEC-6中局灶黏附蛋白的酪氨酸磷酸化和信号复合物的组装，这是一种成熟的恢复模型。1d)表征野生型或显性阴性FAK表达细胞的信号转导和有丝分裂。假设2：在GI肽的作用下，EGFR反激活介导了一种新的促生长信号转导途径。假设2:2a)的目的是确定导致GI肽诱导的EGFR快速反激活的细胞内机制。2b)确定EGFR在GI肽介导的有丝分裂发生信号传导中的作用。2c)表征IEC-6细胞中gpcr诱导的EGFR转激活。

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.