Complex G-protein signaling of the adhesion GPCR, ADGRA3

ADGRA3 (GPR125) is an orphan adhesion G protein-coupled receptor (aGPCR) involved in planar cell polarity, primarily through recruitment of the signaling components disheveled (DVL) during vertebrate gastrulation and discs large homolog 1, implicated in cancer. Limited knowledge exists of the canonical G protein-coupled receptor pathways downstream of ADGRA3. Here, we employed a series of human cell line-based signaling assays to gain insight into the G protein-mediated signaling of ADGRA3. We designed ADGRA3 constructs based on transcript variant analysis in publicly available human liver and brain RNA-seq datasets. Cleavage in the GPCR autoproteolysis site (GPS) is an aGPCR hallmark; thus, we generated a truncated ADGRA3 (C-terminal fragment, CTF) corresponding to a potential cleavage at the GPS. We found low-level activation of Gi and Gs by ADGRA3 and slightly more by its CTF. As the N terminus of the CTF constitutes a class-defined tethered agonist (so-called stachel peptide), we removed the initial three amino acids of the CTF. This resulted in abrogated G protein-mediated signaling, as observed for other aGPCRs. Due to the central role of ADGRA3 in planar cell polarity signaling through DVL recruitment, we investigated the G-protein signaling in the absence of DVL1-3 and found it sustained. No transcriptional activation was observed in an assay of downstream β-catenin activity. Collectively, this establishes classical G protein-mediated signaling for ADGRA3.