Evaluation of CD97 signaling in transgenic mice

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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.