GI PEPTIDE SIGNALING THROUGH TYROSINE PHOSPHORYLATION

描述（由申请人提供）：许多胃肠道（GI）肽、神经递质和生物活性脂质与G蛋白偶联受体（GPCR）结合，并诱导其靶细胞中的细胞迁移和增殖。这些过程在胃肠道粘膜的许多形式的损伤的修复中起重要作用，包括糜烂、溃疡、炎性肠病、肠病性感染、局部缺血和辐射。该提案的广泛，长期目标是阐明介导GPCR诱导的肠上皮细胞迁移和增殖的信号转导途径。除了通过经典的第二信使（例如，Ca 2+，DAG，cAMP），一个新出现的主题是GPCR激动剂还诱导多种蛋白质的酪氨酸磷酸化，包括非受体酪氨酸激酶p125粘着斑激酶（FAK）和衔接蛋白p130 Crk相关底物（CAS）和桩蛋白，这些蛋白质与细胞迁移和增殖有关。GPCR刺激还诱导快速表皮生长因子受体（EGFR）反式激活。在过去的资助期间，我们的研究确定，GPCR激动剂，包括调节肽和生物活性脂质诱导酪氨酸磷酸化级联反应，涉及FAK，Src，Pyk 2和EGFR在各种细胞，包括肠上皮细胞。这些研究为进一步探索酪氨酸磷酸化事件促进GPCR刺激的细胞迁移和增殖的机制奠定了基础。基于我们以前的研究结果，我们的中心假设是酪氨酸磷酸化级联在GPCR诱导的肠上皮细胞的细胞迁移和有丝分裂中起关键作用。本提案将遵循以下具体假设和目标：GPCR激动剂诱导参与细胞迁移和细胞周期进程调节的粘着斑酪氨酸激酶的酪氨酸和丝氨酸磷酸化。源自假设I的目的：1）表征IEC-18和T84肠上皮细胞中响应于GPCR激动剂和EGFR配体的FAK的新磷酸化位点的调节。2)阐明FAK在肠上皮细胞迁移、DNA合成、增殖和信号转导中的作用。假设二：EGFR反式激活有助于肠上皮细胞中GPCR激动剂的促有丝分裂信号传导。3）表征在肠上皮细胞中介导GPCR诱导的EGFR反式激活的信号转导途径，并确定该过程在这些细胞中GPCR诱导的有丝分裂发生中的作用。介导的肠上皮细胞有丝分裂发生。

DESCRIPTION (provided by applicant): Many gastrointestinal (Gl) peptides, neurotransmitters and bioactive lipids bind to G protein-coupled receptors (GPCRs) and induce cell migration and proliferation in their target cells. These processes play an essential role in the repair of many forms of injury to the mucosa of the Gl tract, including erosions, ulcers, inflammatory bowel disease, enteropathic infections, ischemia and radiation. The broad, long-term objective of this proposal is to elucidate the signal transduction pathways that mediate GPCR-induced migration and proliferation in intestinal epithelial cells. In addition to signaling via classic second messengers (e.g., Ca2+, DAG, cAMP), an emerging theme is that GPCR agonists also induce tyrosine phosphorylation of multiple proteins, including the non-receptor tyrosine kinase p125 focal adhesion kinase (FAK) and the adaptor proteins p130 Crk-associated substrate (CAS) and paxillin, which are implicated in cell migration and proliferation. GPCR stimulation also induces rapid epidermal growth factor receptor (EGFR) transactivation. During the past funding period, our studies established that GPCR agonists, including regulatory peptides and bioactive lipids induce tyrosine phosphorylation cascades involving FAK, Src, Pyk2 and EGFR in a variety of cells, including intestinal epithelial cells. These studies form the basis for further exploration of the mechanisms by which tyrosine phosphorylation events contribute to cell migration and proliferation in GPCR-stimulated cells described in the current grant proposal. Based on our previous findings, our central hypothesis is that tyrosine phosphorylation cascades play a critical role in GPCR-induced cellular migration and mitogenesis in intestinal epithelial cells. This proposal will pursue the following specific hypotheses and aims: Hypothesis I. GPCR agonists induce tyrosine and serine phosphorylation of focal adhesion tyrosine kinases implicated in the regulation of cell migration and cell cycle progression. Aims emanating from hypothesis I: 1) Characterize the regulation of novel phosphorylation sites of FAK in response to GPCR agonists and EGFR ligands in IEC-18 and T84 intestinal epithelial cells. 2) Define the role of FAK in mediating migration, DNA synthesis, proliferation and signal transduction in intestinal epithelial cells. Hypothesis II: EGFR transactivation contributes to transduce mitogenic signaling by GPCR agonists in intestinal epithelial cells Aims emanating from hypothesis II: 3) Characterize the signal transduction pathways that mediate GPCR-induced EGFR transactivation in intestinal epithelial cells and determine the role of this process in GPCR-induced mitogenesis in these cells 4) Define the signaling mechanism(s) by which EGFR transactivation contributes to GPCR-mediated mitogenesis in intestinal epithelial cells.