Moreno-Salinas Ana L, Mancini Arturo, Aouad Samya, Kandasamy Herthana, Morissette Sandra, Maiga Arhamatoulaye, Bouvier Michel, Leduc Richard, Sabbagh Laurent
Department of Pharmacology-Physiology, Université de Sherbrooke, Sherbrooke, QC J1H 5N4, Canada.
Domain Therapeutics North America Inc., Montreal, QC H4S 1Z9, Canada.
Cells. 2025 Aug 19;14(16):1284. doi: 10.3390/cells14161284.
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.
黏附G蛋白偶联受体ADGRE5/CD97在许多癌症中上调，是肿瘤学/免疫肿瘤学中的一个潜在药物靶点。然而，ADGRE5的激活和信号传导机制仍知之甚少。在此，我们使用基于增强型旁观者生物发光共振能量转移（ebBRET）的生物传感器和三种策略来表征人（h）ADGRE5信号传导。首先，一种可被烟草蚀纹病毒（TEV）蛋白酶切割的合成受体嵌合体，能够在GPCR蛋白水解位点（GPS）进行可控的拴系激动剂（TA）暴露，揭示了通过Gα12和Gα13的信号传导，以及β-抑制蛋白1/2（β-Arrs）的募集。其次，我们研究了牙龈蛋白酶K（Kgp）引发的野生型hADGRE5信号传导，Kgp是一种在内肽酶，可在GAIN结构域上游切割hADGRE5。Kgp反映了TEV诱导的信号传导，但也促进了Gαz和Gα11的活性。hADGRE5的GPS缺失并不阻断Kgp诱导的受体激活，揭示了一种不依赖GPS切割的作用机制。最后，我们开发了一种检测方法，以β-Arr2作为读出指标来研究hADGRE5的机械刺激（MS）。MS促进了表达hADGRE5的细胞中β-Arr2的募集，而这种反应在GPS缺失后消失。针对hADGRE5配体CD55的中和抗体显著减弱了MS诱导的β-Arr2结合。总体而言，这项研究推进了我们对hADGRE5信号传导的理解，并突出了该受体通过依赖和不依赖GPS切割的机制激活途径的可塑性。
