Biased Signaling 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 release 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 signaling bias directed at potentiating the activity of select G proteins such as G i2 and G 12/13 . These data unveil the underpinnings of biased signaling for aGPCRs defined by GPS autoproteolysis.