Disentangling autoproteolytic cleavage from tethered agonist–dependent activation of the adhesion receptor ADGRL3

Biased Signaling is Structurally Encoded As An Autoproteolysis Event in Adhesion G Protein-Coupled Receptor Latrophilin-3/ADGRL3

“…The productivity aspect is lacking in our approach since the goal of this study was to look at direct G protein coupling dynamics, thus downstream effectors were not assayed in parallel experiments giving the many limitations emanating from monitoring multiple convergent and divergent pathways due to ADGRL3 promiscuous coupling with many G proteins but also with G protein-independent signaling pathways 5,8,17,22,31 . On one hand, our data on G12/13 potentiation conceptually depart from other studies which reported either no changes or a significant decrease in the activity of a reporter biosensor bearing a promoter sensitive to G12/13 downstream signaling induced by an ADGRL3 cleavagedeficient mutant 19 . However, on the other hand, our data on unaffected Gs coupling support the effect of the same cleavage-deficient mutant on cAMP-dependent synapse formation events 5 .…”

“…Thus, we opted to generate a T855G mutation effectively abrogating the nucleophilic potential thought to be essential for bond breakage between the Leucine and Threonine residues as part of the CNHL P1 -T P1' motif that would normally create a free N-termini at the P1' position, located immediately downstream of cleavage (Figure 1A). A similar modification engineered into ADGRL3 mouse homolog was recently shown to result in a cleavage-deficient mutant receptor that maintained an intact TA activity 19 . Analysis of total cell lysates from receptor-expressing HEK293 cells using immunoblotting of NTFand CTF-associated epitopes, FLAG and HA tag respectively, yielded signals as single bands for both ADGRL3-WT and T855G mutant receptor evidencing proper expression.…”

“…Receptor activation protocols relied on the intrinsic constitutive activity described for ADGRL3. Different levels of constitutive activity were generated by co-transfecting increasing DNA amounts of each receptor separately with BRET-based G protein biosensors, therefore leading to increasing receptor expression and consequently promoting the formation of a higher number of active G protein-receptor complexes 8,19 .…”

“…given by the tethered agonist present in aGPCRs due to GPS cleavage, we sought to investigate the role of autoproteolysis in modulating intrinsic receptor coupling to G proteins. Although previous studies have reported the impact of GPS cleavage on downstream effectors or second messengers, none had addressed the dynamics of direct receptor coupling to its upstream effectors, the G proteins 5,19 . Using a profiling strategy involving BRET-based biosensors of different G protein families, we provide evidence that GPS cleavage supports an intrinsically biased agonism towards specific G proteins within ADGRL3.…”

The adhesion GPCRs CELSR1-3 and LPHN3 engage G proteins via distinct activation mechanisms

“…This approach is based on the entirely P'-substrate selectivity of enterokinase, which cleaves following DDDK/ 54 . This system has been used to generate functional native exposed TAs for ADGRG6 54 and LPHN3 61 . We adopted this approach for CELSR2 by capping the native CELSR2 TA (N-TSFAVLM…-C) with SNAP tag-linker-FLAG (Figure 7D).…”