Use of BRET‐based biosensors to characterize the adhesion GPCR ADGRE5/CD97 signalling profile
Authors: Maiga Arhama , Simard Elie , Leduc Richard , Bouvier Michel 
Published: June 2021

ADGRE5 or CD97 is an adhesion GPCR broadly expressed in normal hematopoietic cells, smooth muscle cells and has been proposed to play important roles during development. Its expression is found to be increased in a growing number of cancers including acute myeloid leukemia, prostate, gastric, pancreatic, rectal and gallbladder cancers.The receptor is expressed as three alternatively spliced isoforms characterized by the presence of three to five EGF like motifs that play important roles in the binding of differents cells partners, including integrins, CD55 and chondroitin sulfate B. Interstingly, despite their ability to bind to the receptor and participate to its adhesion function, none of these extra‐cellular ligands were linked to signalling activity downstream of the receptor. The lack of functional agonist makes CD97 characterization difficult and very little is known about the downstream signalling pathway(s) that can be engaged by the receptor. A constitutive ability of CD97 towards the G12/G13‐Rho pathway has been reported upon overexpression of the receptor using a gene reporter system[1].Recent studies on some adhesion GPCRs have demonstrated that the dissociation of their extra cellular domain (ECD) up to their GPCR proteolysis site (GPS) is important for their activation since it reveals a tethered ligand that can activate some of the aGPCR. Consistent with this mode of activation, it has been shown that N‐terminal truncated forms up to the GPS have higher constitutive activity than their wild type (WT) counterparts. Also, soluble peptides mimicking the tethered ligands were shown to activate aGPCRs such as GPR126, GPR133, GPR56 and GPR110 [2 3].The aim of the study was to characterize the signalling activity of ADGRE5/CD97 and to assess whether the thetered ligand mode of activation also applies to this member of the adhesion GPCR family. For this purpose we used a collection of BRET‐based biosensors monitoring the real time activation of G12, G13, Gs, Gq and b‐arrestin in living cells. The constitutive activity of the WT and GPS‐truncated (CD97‐GPS) forms of CD97 were assessed on each pathways by expressing increasing concentration of the receptor. No significant constutive activity was observed for any of the pathways tested upon overexpression of the wt receptor whereas constitutive activation of all the pathways tested was observed following expression of CD97‐GPS. Signalling downstream of G12/13 was confirmed by monitoring the recruitment of Rho‐GEF‐P115 to the plasma membrane upon over‐expression of CD97‐GPS. Sequential deletion of amino acids from the GPS site into the proposed thetered ligands resulted in a reduced constitutive activation, suggesting the presence of a cryptic activating peptide in the N‐terminus of CD97. To further test this hypothesis, synthetic peptides mimicking the putative the thered ligands were tested for ther ability to activate G13 and b‐arrestin. Our prelimirary results suggest that such a 13 amino acid peptide may activate the receptor.Taken together our data suggest that CD97 can engage G protein and b‐arrestin signalling pathways and that the activation may involve a thetered agonist revealed by the cleavage of CD97 at the GPS. The assays developed in the present study are suitable for hight throughtup screening for the identification of synthetic agonists and antagonists for the study of this receptor.Support or Funding InformationMITACS, Domain therapeuticsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.