Biased signalling is structurally encoded as an autoproteolysis event in adhesion G protein-coupled receptor Latrophilin-3/ADGRL3.

Adhesion G protein-coupled receptors (aGPCRs) possess a unique topology, including the presence of a GPCR proteolysis site (GPS), which, upon autoproteolysis, generates two functionally distinct fragments that remain non-covalently associated at the plasma membrane. A proposed activation mechanism for aGPCRs involves the exposure of a tethered agonist, which depends on cleavage at the GPS. However, this hypothesis has been challenged by the observation that non-cleavable aGPCRs exhibit constitutive activity, thus making the function of GPS cleavage widely enigmatic. In this study, we sought to elucidate the function of GPS-mediated cleavage through the study of G protein coupling with Latrophilin-3/ADGRL3, a prototypical aGPCR involved in synapse formation and function. Using BRET-based G protein biosensors, we reveal that an autoproteolysis-deficient mutant of ADGRL3 retains constitutive activity. Surprisingly, we uncover that cleavage deficiency leads to a signalling bias directed at potentiating the activity of select G proteins such as Gi2 and G12/13. These data unveil the underpinnings of biased signalling for aGPCRs defined by GPS autoproteolysis.