Evaluation of CD97 signaling in transgenic mice

转基因小鼠 CD97 信号传导的评估

• 批准号: 266144110
• 负责人: Professorin Dr. Gabriela Aust
• 金额: --
• 依托单位: Forschungslaboratorien der Chirurgischen Kliniken I und II
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/266144110?language=en
• 关键词: Evaluation; CD97; signaling; transgenic; mice

CD97, as a prototypic adhesion GPCR (aGPCR), is cleaved autocatalytically at its GPCR proteolysis site (GPS) into the N- (NTF) and C-terminal fragment (CTF). AGPCR relay potentially several signals either by employing the NTF, CTF, and/or by a NTF-CTF interplay. Additionally, molecular interactions via the PDZ-domain binding motif (PBM) at their C-terminus, present in CD97, facilitate the assembly of intracellular supramolecular complexes. Interestingly, the signaling of CD97 likely depends on the regulated subcellular residence of the receptor, which locates within lateral cell contacts of normal intestinal epithelial cells but intracellularly and at the leading edge of the corresponding (migrating) tumor cells. This indicates context-dependent CD97 functions, which are based on specific molecular signal pathways and interaction partners. Project P8 will address the following points: (1) Determination of the structural requirements for CD97 signaling. Various transgenic mice strains expressing CD97 variants in intestinal epithelial cells combined with robust in vivo and in vitro functional read-outs are suitable to address this question. We will investigate the role of the NTF and the PBM, binding to the extracellular ligand Thy1, and cleavage at the GPS in new Cd97-transgenic mice. (2) The N-terminal extracellular stretch of CTF (= stachel peptide) probably acts as a tethered agonist in aGPCR signaling. We will clarify whether CD97 could be specifically activated by the tethered stachel peptide and whether this activation depends on the presence of lysophosphatidic acid (LPA)-receptors which heterodimerize with CD97. (3) Identification of the intracellular interaction partners of CD97 will help to link it to signaling cascades and molecular networks. Tandem affinity purification (TAP) will be conducted to address how the CD97 interactome looks like. The role of phosphorylation at the PBM for the contextdependent change in CD97 function will be verified. Thus, the long-term aim of this project is to evaluate and verify signaling models for the CD97 NTF, CTF and NTF-CTF interplay and to characterize the intracellular interaction and signaling partners of CD97. This will allow new insights into common and CD97-specific functions and signaling routes of this receptor.

CD97 作为一种原型粘附 GPCR (aGPCR)，在其 GPCR 蛋白水解位点 (GPS) 处被自催化裂解为 N 端 (NTF) 和 C 端片段 (CTF)。 AGPCR 通过使用 NTF、CTF 和/或通过 NTF-CTF 相互作用来中继潜在的多个信号。此外，CD97 中存在的通过 C 端 PDZ 结构域结合基序 (PBM) 进行的分子相互作用促进了细胞内超分子复合物的组装。有趣的是，CD97 的信号传导可能取决于受体受调节的亚细胞驻留，该受体位于正常肠上皮细胞的侧细胞接触内，但位于细胞内和相应（迁移）肿瘤细胞的前缘。这表明 CD97 功能具有环境依赖性，其基于特定的分子信号通路和相互作用伙伴。 P8项目将解决以下几点： (1)确定CD97信令的结构要求。在肠上皮细胞中表达 CD97 变体的各种转基因小鼠品系与强大的体内和体外功能读数相结合，适合解决这个问题。我们将研究 NTF 和 PBM 的作用、与细胞外配体 Thy1 的结合以及新 Cd97 转基因小鼠中 GPS 的切割。 (2) CTF（= stachel 肽）的 N 端胞外段可能在 aGPCR 信号传导中充当束缚激动剂。我们将阐明 CD97 是否可以被束缚的 stachel 肽特异性激活，以及这种激活是否取决于与 CD97 异二聚化的溶血磷脂酸 (LPA) 受体的存在。 (3) CD97 细胞内相互作用伙伴的鉴定将有助于将其与信号级联和分子网络联系起来。将进行串联亲和纯化 (TAP) 以解决 CD97 相互作用组的问题。将验证 PBM 磷酸化对于 CD97 功能上下文依赖性变化的作用。因此，该项目的长期目标是评估和验证 CD97 NTF、CTF 和 NTF-CTF 相互作用的信号传导模型，并表征 CD97 的细胞内相互作用和信号传导伙伴。这将使人们对该受体的常见功能和 CD97 特异性功能以及信号传导途径有新的认识。