Complex G-protein signaling of the adhesion GPCR, ADGRA3.

ADGRA3 (GPR125) is an orphan adhesion G protein-coupled receptor (aGPCR) that plays a role in planar cell polarity (PCP), primarily through recruitment of the signaling components Dishevelled (DVL) during vertebrate gastrulation, and Discs large homolog 1 (Dlg1), which is implicated in cancer. Limited knowledge exists of the signaling capacity in the canonical GPCR pathways of ADGRA3. Here, we employed a series of human cell line-based signaling assays to gain insight into the heterotrimeric G protein-mediated signaling of ADGRA3. We based the design of ADGRA3 constructs on analyses of transcript variants in publicly available human liver and brain RNA-seq datasets. As cleavage in the GPCR autoproteolysis site (GPS) is a hallmark for many aGPCRs, 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 known as the 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 PCP signaling through DVL recruitment, we investigated the G-protein signaling in absence of DVL1-3 and found it sustained. No transcriptional activation was observed downstream of β-catenin in an assay reporting T-cell factor/lymphoid enhancer factor (TCF/LEF)-mediated transcriptional activity. Collectively, this establishes a classical G protein-mediated signaling for ADGRA3 in addition to its association with components of non-canonical Wnt-signaling pathways.