ADGRA3 is a self-activated, Gi-coupled adhesion GPCR expressed in liver progenitor cells

ADGRA3 (GPR125) is an orphan adhesion GPCR (aGPCR) expressed in the murine liver and kidney. Like other aGPCRs, the large N-terminal fragment (NTF) of ADGRA3 contains a GPCR autoproteolysis-inducing (GAIN) domain, but the cleavage site (GPS) differs from that of other aGPCRs (SL/S in ADGRA3 vs. HL/S in most aGPCRs). To confirm that ADGRA3 can be cleaved, we cloned the full-length receptor (Adgra3FL) and a variant of Adgra3 lacking the GPS (Adgra3AAA) with N-terminal Flag and C-terminal HA tags. Western blot analyses detected the full-length receptor as well as the C-terminal fragment (CTF, which remains following cleavage and removal of the NTF) in HEK293T cells expressing ADGRA3FL and ADGRA3AAA, suggesting that, while ADGRA3 is cleaved, the cleavage site might differ from the predicted GPS. Removal of the NTF from typical aGPCRs uncovers a tethered agonist; to determine if ADGRA3 signals through a similar manner, we used the PRESTO-TANGO assay, which can detect receptor activation independent of G-signaling pathway. ADGRA3FL and ADGRA3CTF demonstrated activity in the absence of any exogenous ligands, suggesting that ADGRA3 can self-activate, most likely through the β-strand tethered ligand (B13) immediately following the predicted GPS. Indeed, ADGRA3FL and ADGRA3CTF exhibited greater activity following exposure to synthetic peptides that correspond to the B13 tethered agonist. We also confirmed previous reports that ADGRA3 can be activated by the exogenous ligand hesperetin. Previous studies have suggested that ADGRA3 can signal via both Gαs and Gαi pathways. To assess ADGRA3 signaling, we used the Dual Luciferase assay, a cAMP reporter assay that can distinguish between Gαs and Gαi signaling. We found that activation of ADGRA3 with either hesperetin or synthetic B13 peptides led to a decrease in forskolin-elevated cAMP levels, but no changes in cAMP levels were detected in the absence of forskolin. Additionally, treatment with pertussis toxin, a potent Gαi inhibitor, diminished receptor activity relative to non-treated samples. To confirm G-protein coupling to ADGRA3, we performed a bioluminescence resonance energy transfer (BRET) assay. We found that ADGRA3 couples with Gαi2, but not Gαs. These findings imply that ADGRA3 signals specifically via a Gαi pathway. To unpack the function of ADGRA3, we first analyzed ADGRA3 localization in vivo using RNAscope and found robust expression of Adgra3 in the cuboidal endothelial cells lining the bile duct, as well as generalized expression in hepatocytes. To confirm these expression patterns, we crossed an Adgra3 knock-out mouse (Adgra3cre), where exon 1 of Adgra3 is replaced with an inducible Cre recombinase, to a conditional reporter mouse (Rosa-tdTomato). We found the most robust expression in a subset of hepatocytes reminiscent of hepatic progenitor cells. Lastly, we investigated the physiological role of ADGRA3 in the liver and kidney by measuring body weight, glomerular filtration rate, glucose tolerance, and hepatic AST/ALT levels. We found no significant differences between KO and WT adult mice. Future work is ongoing to determine if ADGRA3 contributes to liver repair following liver damage induced by a choline-deficient diet. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.