Domain-specific N-glycosylation of the adhesion G-protein-coupled receptor ADGRG6 N-terminal fragment regulates trafficking, proteolytic processing, and signaling.

Adhesion G-protein-coupled receptors (aGPCRs) are characterized by long extracellular N-terminus fragments (NTF) with several adhesive domains. Many aGPCRs are cleaved at the GPCR-autoproteolysis site (GPS), enclosed within the larger GPCR-autoproteolysis-inducing (GAIN) domain. Following cleavage at the endoplasmic reticulum (ER), the NTF and C-terminal fragments (CTF) heterodimerize, and the protomer is trafficked to the plasma membrane. ADGRG6 is an aGPCR playing important roles in embryonic development. It is activated by mechanical perturbation of the NTF and several extracellular matrix (ECM) proteins. GPCRs are known to undergo several posttranslational modifications (PTM) that regulate the molecular pharmacology of these receptors. N-glycosylation is an important PTM that regulates GPCR expression, trafficking, ligand binding, and signaling bias. Although ADGRG6 is N-glycosylated, the location of the glycans remains unknown. Furthermore, are there spatial roles of N-glycosylation in ADGRG6 processing, trafficking, and signaling? To address these gaps in knowledge, we used biochemical and cell-biological approaches using cell lines overexpressing wild-type and N-glycosylation mutants of ADGRG6. We demonstrate that specific N-glycan residues in different domains of the NTF of ADGRG6 have distinct roles in ADGRG6 autoproteolysis, furin cleavage, trafficking to the plasma membrane, and cAMP production.