N-terminal fragment shedding contributes to signaling of the full-length adhesion receptor ADGRL3

Most adhesion G protein-coupled receptors (GPCRs) undergo autoproteolytic cleavage during receptor biosynthesis, resulting in noncovalently bound N-terminal fragments (NTFs) and C-terminal fragments (CTFs) that remain associated during receptor trafficking to the plasma membrane. While substantial evidence supports increased G protein signaling when just the CTF is expressed, there is an ongoing debate about whether NTF removal is required to initiate signaling in the context of the WT receptor. Here, we use adhesion GPCR latrophilin-3 (ADGRL3) as a model receptor to investigate tethered agonist (TA)-mediated activation. First, we show that extending the N terminus of the TA in ADGRL3 CTF disrupts G protein signaling. This suggests that if the TA is not fully exposed, it is unlikely to interact with the orthosteric pocket in an optimal manner for G protein activation. Second, we show that when full-length ADGRL3 is expressed in heterologous cells, approximately 5% of the receptor population spontaneously sheds its NTF. We hypothesized that the signaling activity observed for full-length ADGRL3 is largely because of this shedding, which exposes the native TA. To test this hypothesis, we used a full-length cleavage-deficient ADGRL3 mutant. Compared with WT receptor, this mutant lost ∼80% of its signaling through Gα₁₃ and showed a much lower level of spontaneous NTF shedding, approximately 20% of that observed for WT receptor. This loss of spontaneous NTF shedding likely explains its diminished signaling activity. These findings suggest that TA-mediated signal transduction by full-length ADGRL3 requires removal of its NTF.