Molecular studies of ADGRG6 suggest its extracellular CUB domain is a key regulator of cAMP signaling

Abstract ID 99823 Poster Board 360 ADGRG6 is a member of the adhesion G Protein-coupled receptor (aGPCR) family that regulates Schwann cell myelination among other physiologic processes through its multidomain extracellular region (ECR). In addition to mediating developmental functions, the ECR of ADGRG6 is found mutated in several cancers and other diseases. While it has been shown that closed and open ECR conformations relate to low and high ADGRG6 signaling levels respectively, the mechanism of this regulation remains poorly understood. Furthermore, the extracellular Complement C1r/C1s, Uegf, Bmp1 (CUB) domain in ADGRG6 mediates this closed conformation and potentially binds to ligands, but a direct relationship between these functions and ADGRG6 signaling is lacking. Here, we show that deletion of the CUB domain in zebrafish and human ADGRG6 decreases the basal intracellular cAMP signaling levels of the WT receptor. Using molecular dynamics (MD), we simulated several point mutations sourced from clinical databases that are found at interdomain contact points between CUB and other ECR domains. We identified a CUB domain mutation (C94Y) which exhibited a completely open conformation in MD simulations by breaking interactions between CUB and the rest of the ECR. In cellular signaling assays, ADGRG6 C94Y caused significantly higher levels of cAMP than the WT receptor, suggesting that these interactions regulate ECR signaling. A second mutation (Y96A) in the putative ligand binding site of the CUB domain inhibited the receptor's capacity to elevate intracellular cAMP levels. Taken together, these experiments suggest that the CUB domain of ADGRG6 regulates cAMP signaling by adopting different conformations and possibly binding to an extracellular ligand.