Self-cleavage of the GAIN domain of adhesion G protein-coupled receptors requires multiple domain-extrinsic factors.

The autoproteolysis-inducing (GAIN) domain of class B2/adhesion G protein-coupled receptors (aGPCRs) is structurally conserved, and its self-cleavage is central to receptor mechanotransduction and signaling. Yet, the influence of factors beyond the protein fold on GAIN domain autoproteolysis remains unclear. Using ADGRE2/EMR2, a self-cleaved aGPCR, we investigated contributions of the seven-transmembrane (7TM) region to GAIN domain autoproteolysis during receptor maturation and trafficking. Retention Upon Selective Hook (RUSH) assays showed that self-cleavage acts as a checkpoint before endoplasmic reticulum (ER) exit, but not during plasma membrane transport. Stepwise truncations of the 7TM domain revealed that cleavage can occur before or at synthesis of the first transmembrane helix, and is enhanced with formation of the full 7TM domain. Analyses of six additional cleavage-competent aGPCRs demonstrated that ER membrane tethering facilitates GAIN domain processing, supported by proteomic evidence linking cleavage to proximity with the N-glycosylation pathway. These results highlight the interplay between GAIN and 7TM domains, offering mechanistic insights and guiding pharmacological strategies to modulate aGPCR activation and signaling.