ADGRA3: In vivo hepatic localization, functional importance, and in vitro activation

G protein-coupled receptors (GPCRs), which encode for more than 3% of all human genes, are the largest protein family in the human genome. However, most GPCRs remain under-characterized with little known about their expression, agonists, and physiological function. To identify understudied receptors in the liver, the largest metabolic organ in the body, we performed a TaqMan array screen of more than 300 GPCRs in both male and female C57BL6 murine livers. Among the most highly expressed orphan receptors is ADGRA3 (GPR125), an adhesion GPCR (aGPCR), whose expression is comparable to that of the glucagon receptor, with ΔCt values of 17.2 and 16.0 respectively. aGPCRs possess a unique N-terminal domain that includes a GPCR auto proteolysis-inducing (GAIN) domain, which contains a GPS cleavage site that allows for the cleavage and non-covalent attachment of the N-Terminal (NTF) and C-Terminal Fragments. It is theorized that the displacement of the NTF exposes a tethered ligand, which ultimately results in ADGRA3 activation. ADGRA3 has an unconventional GPS site and it was unclear if this receptor follows a similar activation sequence. To test this, we synthesized peptides corresponding to the β-strand immediately following the predicted GPS domain and tested for activation using the PRESTO-TANGO assay, a luminescence readout to indicate receptor activation. We confirmed that ADGRA3 is activated by a tethered ligand, observing maximal activation with the complete β-strand and 2 additional C-Terminal amino acids. We then further employed the PRESTO-TANGO assay to deorphanize ADGRA3 by identifying non-self activators in a large, unbiased chemical screen (>500 chemicals). This resulted in the identification of more than 25 unique ligands whose selective binding results in ADGRA3 activation. To date, our studies show that ADGRA3 is best activated by C16H20N2O3. By using RNA scope, we localized Adgra3 transcripts to the cuboidal endothelial cells in the lining of the bile duct and hepatocytes. This localization was confirmed through antibody staining, which revealed that ADGRA3 localizes to the basolateral membrane of cholangiocytes. To investigate physiological function of hepatic ADGRA3, we are characterizing the C57BL6-ADGRA3-GreenLantern mouse (GL-KO), where insertion of a GreenLantern tag within ADGRA3 is predicted to disrupt functionality. To visualize the impacts of ADGRA3 disruption, we employed functional Magnetic Resonance Imaging (MRI) to track the uptake of gadoxetate disodium (EOVIST). Injection of exogenous EOVIST is selectively taken up by the liver through organic anion transporting polypeptides (OATP) and is further cleared into the gallbladder. In wildtype ADGRA3 mice, EOVIST was rapidly taken up by the liver and was found within the common bile duct entering the gallbladder by 60 minutes. In the GL-KO, this clearance was delayed with minimal accumulation in the common bile duct. Given the localization of ADGRA3 within the cholangiocytes, this raises the possibility that ADGRA3 may play an essential role in hepatic development and biliary repair mechanisms. NIH/NCATS. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.