Decoding ADGRE5: How Proteolytic Cleavage and Mechanical Forces Unleash Cellular Signals

The adhesion G protein-coupled receptor ADGRE5/CD97 is upregulated in many cancers, representing a potential drug target in oncology/immuno-oncology. Yet, ADGRE5's activation and signaling mechanisms remain poorly understood. Here, we used enhanced bystander bioluminescence resonance energy transfer (ebBRET)-based biosensors and three strategies to characterize human (h) ADGRE5 signaling. First, a synthetic tobacco etch virus (TEV) protease-cleavable receptor chimera enabling controlled tethered agonist (TA) exposure at the GPCR proteolysis site (GPS) revealed signaling through Gα12 and Gα13, along with the recruitment of β-Arrestins 1/2 (β-Arrs). Second, we investigated WT hADGRE5 signaling elicited by Gingipain K (Kgp), an endopeptidase that cleaves hADGRE5 upstream of the GAIN domain. Kgp mirrored TEV-induced signaling but also promoted Gαz and Gα11 activity. The abolition of hADGRE5's GPS did not block Kgp-induced receptor activation, revealing a GPS cleavage-independent mechanism of action. Finally, we developed an assay to study hADGRE5 mechanical stimulation (MS) using β-Arr2 as a readout. MS promoted β-Arr2 recruitment in hADGRE5-expressing cells, and this response was lost upon abolition of the GPS. A neutralizing antibody to the hADGRE5 ligand CD55 significantly dampened MS-induced β-Arr2 engagement. Overall, this study advances our understanding of hADGRE5's signaling and highlights the receptor's plasticity in activating pathways via both GPS cleavage-dependent and -independent mechanisms.