Evaluation of CD97 signaling in transgenic mice

作为原型粘附GPCR（aGPCR），CD 97在其GPCR蛋白水解位点（GPS）处被自催化切割成N-（NTF）和C-末端片段（CTF）。AGPCR通过采用NTF、CTF和/或通过NTF-CTF相互作用潜在地中继若干信号。此外，分子间的相互作用通过PDZ结构域结合基序（PBM）在其C-末端，存在于CD 97，促进组装的细胞内超分子复合物。有趣的是，CD 97的信号传导可能取决于受体的调节亚细胞驻留，其位于正常肠上皮细胞的侧向细胞接触内，但位于细胞内和相应（迁移）肿瘤细胞的前缘。这表明背景依赖性CD 97功能，其基于特定的分子信号通路和相互作用伴侣。项目P8将解决以下几点：（1）确定CD 97信号传导的结构要求。在肠上皮细胞中表达CD 97变体的各种转基因小鼠品系与稳健的体内和体外功能读数组合适合于解决这个问题。我们将研究NTF和PBM的作用，结合到细胞外配体Thy 1，并在新的Cd 97转基因小鼠的GPS裂解。(2)CTF（= stachel肽）的N-末端胞外段可能在aGPCR信号传导中充当系留激动剂。我们将阐明CD 97是否可以被拴系的stachel肽特异性激活，以及这种激活是否依赖于与CD 97异源二聚化的溶血磷脂酸（LPA）受体的存在。(3)鉴定CD 97的细胞内相互作用伙伴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CD 97相互作用组的外观。将验证PBM磷酸化对CD 97功能的上下文依赖性变化的作用。因此，本项目的长期目标是评估和验证CD 97 NTF、CTF和NTF-CTF相互作用的信号模型，并表征CD 97的细胞内相互作用和信号伴侣。这将使新的见解共同和CD 97特异性功能和信号通路的这种受体。

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.