ADGRA3 Is a Self-activated Adhesion GPCR Expressed in the Kidney and Liver

While G protein-coupled receptors (GPCRs) comprise the largest protein family in the human genome, the expression patterns, activation and physiological function of most GPCRs remain unknown. Using a TaqMan array screen of more than 300 GPCRs, we identified Adgra3 (GPR125) as a highly expressed orphan GPCR in murine liver (ΔC q value of 17.2 compared to 16.0 for the glucagon receptor). Like other adhesion GPCRs (aGPCRs), the large N-terminal fragment (NTF) of ADGRA3 contains a GPCR autoproteolysis-inducing (GAIN) domain, but the GPS cleavage site differs from that of other aGPCRs. Therefore, it is unclear whether ADGRA3 undergoes autoproteolysis and self-activation like most aGPCRs. We hypothesized that, despite its atypical GPS site, ADGRA3 is also cleaved and can self-activate through the β-strand immediately following the predicted GPS site. To test this hypothesis, we cloned the full-length ( Adgra3 FL ) and C-terminal fragment of Adgra3 ( Adgra3 CTF , which lacks the NTF, including the GAIN domain, but retains the β-strand) and employed the PRESTO-TANGO and Dual Luciferase assays to assess ADGRA3 activation and its downstream signaling pathway in HEK293T cells. The PRESTO-TANGO assay measures luciferase expression following receptor activation, whereas the Dual Luciferase assay, a cAMP reporter assay, can distinguish between Gɑ s and Gɑ i downstream signaling pathways. We found that ADGRA3 self-activates: ADGRA3 FL demonstrated activity in the absence of any exogenous ligands. ADGRA3 CTF activity was significantly higher than that of ADGRA3 FL , suggesting that the removal of the bulky NTF facilitates receptor activation. To confirm that the β-strand is responsible for this self-activation, we used synthetic peptides corresponding to either the full sequence of the ADGRA3 β-strand (B13) or modified B13 peptides with additional or fewer amino acids. Since the maintenance of the B13 helical structure is critical for ADGRA3 activation, we expected that the modified B13 peptides would not activate ADGRA3. Indeed, we found that only the complete β-strand, but not the modified versions, activates both ADGRA3 FL and ADGRA3 CTF , with significantly higher activation in the latter. Using the Dual Luciferase assay, we detected decreases in forskolin-elevated cAMP levels following both ADGRA3 FL and ADGRA3 CTF activation. We also identified several exogenous compounds that activate ADGRA3 using an unbiased chemical screen of more than 500 chemicals. Taken together, our findings suggest that ADGRA3 is cleaved, can self-activate through its β-strand, and signals through a Gɑ i pathway. To unpack the physiological function of ADGRA3, we used RNAscope to localize Adgra3 expression in the murine liver. We found robust expression of Adgra3 in the cuboidal endothelial cells that line the bile duct, as well as generalized expression in hepatocytes. Additionally, antibody staining revealed that ADGRA3 localizes to the basolateral membrane of cholangiocytes. Efforts are underway to confirm the localization in vivo using an Adgra3 knock-in/knock-out mouse strain ( Adgra3 cre ) and a tdTomato reporter mouse. Our current focus is on using the Adgra3 cre knock-out mouse to elucidate the function of the receptor in the mouse liver and kidney. NIH This abstract was presented at the American Physiology Summit 2025 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.