Herthana Kandasamy and Arturo Mancini, to present a novel poster at the ASPET 2024
Domain Therapeutics North America will participate in the next ASPET (American Society for Pharmacology and Experimental Therapeutics) congress, come in person and follow Herthana Kandasamy and Arturo Mancini’s poster presentation on the “Early-stage development of an in vitro screening assay to characterize the activation of the human adhesion GPCR ADGRE5.“ on May 20th at the Saint Louis Union Station Hotel, poster board 485 between 5 and 7 pm EST, in Saint Louis, Misssouri !
Dr Arturo Mancini is Scientist at Domain Therapeutics North America.
Herthana Kandasamy is Research associate at Domain Therapeutics North America.
ADGRE5 is a prototypical adhesion GPCR (aGPCR) that is expressed mainly on T lymphocytes, monocytes/macrophages, granulocytes, NK cells (especially CD56bright) and smooth muscle cells. Importantly, ADGRE5 expression is upregulated in many cancers (e.g., gastric/colorectal/pancreatic carcinomas, various leukemias, glioblastoma), whereas the corresponding normal tissues express relatively little or no ADGRE5. Previous studies indicated that ADGRE5 plays an important role in modulating cell adherence/detaching, migration, invasion, and metastasis, making ADGRE5 a potential drug target in oncology/immuno-oncology. Yet, the development of assays enabling the discovery of drugs acting on aGPCRs has been complicated by the lack of functional agonists and the complex multimodal mechanisms believed to govern receptor activation. Current methods rely on artificial methods of activation that preclude the identification of allosteric modulators acting upon ADGRE5’s tractable long extracellular N-terminal fragment.
We describe a novel in vitro enhanced bystander (eb)BRET-based assay allowing to detect the activity of the full-length (native) form of human (h)ADGRE5 via its physiological activation mode (i.e., following mechanical stimulation (MS)). Indeed, subjecting full-length hADGRE5 isoform1- or isoform2-transfected HEK293 to vigorous orbital shaking resulted in a rapid time-dependent recruitment of ßArrestin 2 (ßArr2) to the plasma membrane. Such mechanosensitivity was not observed with a control receptor (mu opioid receptor), despite its ability to engage ßArr2 following ligand stimulation. Interestingly, MS-induced Arr2 engagement was absent with hADGRE5 harboring a mutated GPCR proteolysis site (GPS), which is believed to play an important role in aGPCR activation through the exposure of a tethered agonist following cleavage. Lack of activity was not related to defective cell surface expression of the GPS mutant, as both WT and GPS mutant forms of hADGRE5 displayed comparable surface expression when evaluated by flow cytometry. Similarly, MS provoked a delayed internalization of WT hADGRE5 but not that of the GPS mutant.
CD55/DAF is one of four ligands described to interact with hADGRE5’s adhesive EGF-like folds and has been shown to promote MS-dependent downregulation of leukocyte ADGRE5 in vivo. We showed that CD55/DAF is expressed endogenously by HEK293, leading us to hypothesize that trans-signaling complexes between CD55/DAF and hADGRE5 could mediate the hADGRE5 activity observed in our MS assays. Indeed, addition of a neutralizing anti-CD55 antibody significantly dampened MS-induced hADGRE5 ßArr2 recruitment. Finally, the robustness of the MS ßArr2 recruitment assay was evaluated in 96-well plates and resulted in a Z’ = 0.73, thus rendering this assay compatible for high-throughput screening of compound libraries.
Collectively, our ebBRET-based assay is robust and will help identify and characterize novel therapeutic agents acting on (patho)physiologically relevant mechanisms of ADGRE5 activation through both orthosteric and allosteric receptor sites.