GI PEPTIDE SIGNALING THROUGH TYROSINE PHOSPHORYLATION

描述(申请人提供)：许多胃肠道(G1)肽、神经递质和生物活性脂质与G蛋白偶联受体(GPCRs)结合，并在其靶细胞中诱导细胞迁移和增殖。这些过程在修复多种形式的胃肠道粘膜损伤中起着至关重要的作用，包括侵蚀、溃疡、炎症性肠病、肠道感染、缺血和辐射。这一建议的广泛、长期的目标是阐明介导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(如Ca~(2+)、DAG、cAMP)进行信号传递外，一个新的研究主题是GPCR激动剂还能诱导多种蛋白质的酪氨酸磷酸化，包括与细胞迁移和增殖有关的非受体酪氨酸激酶p125粘着斑激酶(FAK)、适配蛋白p130 Crk相关底物(CAS)和Paxlin。GPCR刺激还可诱导表皮生长因子受体(EGFR)的快速反式激活。在过去的资助期间，我们的研究证实了GPCR激动剂，包括调节肽和生物活性脂类，在包括肠上皮细胞在内的多种细胞中诱导涉及FAK、Src、Pyk2和EGFR的酪氨酸磷酸化级联反应。这些研究为进一步探索酪氨酸磷酸化事件促进细胞迁移和增殖的机制奠定了基础，这些机制在当前的拨款提案中有所描述。基于我们以前的发现，我们的中心假设是酪氨酸磷酸化级联在GPCR诱导的细胞迁移和肠上皮细胞有丝分裂中发挥关键作用。这项提议将追求以下特定的假设和目标：假设I.GPCR激动剂诱导局部黏附酪氨酸激酶的酪氨酸和丝氨酸磷酸化，这与细胞迁移和细胞周期进程的调节有关。目的源于假设I：1)表征在IEC-18和T84肠上皮细胞中对GPCR激动剂和EGFR配体响应的FAK新的磷酸化位点的调节。2)明确FAK在介导肠上皮细胞迁移、DNA合成、增殖和信号转导中的作用。假说II：EGFR反式激活有助于通过GPCR激动剂在肠上皮细胞中转导有丝分裂信号。目的源于假说II：3)表征介导GPCR诱导的肠上皮细胞中EGFR反式激活的信号转导途径，并确定这一过程在这些细胞有丝分裂中的作用4)确定EGFR反式激活促进肠上皮细胞有丝分裂的信号机制(S)。

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.