Step-wise mechanical unfolding and dissociation of the GAIN domains of ADGRG1/GPR56, ADGRL1/Latrophilin-1 and ADGRB3/BAI3: insights into the mechanical activation hypothesis of adhesion G protein-coupled receptors

Abstract

Adhesion G protein-coupled receptors (aGPCRs) are a large family within the superfamily of G protein-coupled receptors involved in various physiological processes. One unique feature of aGPCRs is their long N-terminal extracellular regions (ECRs), which contain adhesive domains and a GPCR autoproteolysis-inducing (GAIN) domain. This GAIN domain promotes autoproteolytic cleavage of aGPCRs into N- and C-terminal fragments (NTF, CTF, respectively) after receptor biosynthesis. aGPCR signaling involves an interplay between the NTF and CTF that can be mechanically activated or modulated. However, how force affects the conformation/structure of the GAIN domain as a central structural element in aGPCR activation remains largely unknown. In this study, we investigated the mechanical stability of the GAIN domains of three aGPCRs from subfamilies B, G and L at a loading rate of 1 pN/s. Our findings demonstrate that the GAIN domains can be destabilized by forces from a few to 20 piconewtons (pN). Specifically, for the autocleaved aGPCRs, ADGRG1/GPR56 and ADGRL1/Latrophilin-1, forces over this range can cause detachment of the GAIN domain from the membrane-proximal Stachel element, which serves as an endogenous tethered agonist to aGPCRs, typically preceded with GAIN domain unfolding. For the non-cleavable aGPCR ADGRB3/BAI3, the GAIN domain undergoes complex mechanical unfolding over a similar force range. We also demonstrate that detachment of the GAIN domain can take place during cell migration, provided that the linkage between aGPCR and extracellular matrix is sufficiently stable. These results suggest that both structural stability of the GAIN domain and NTF/CTF dissociation are sensitive to physiological ranges of tensile forces, providing insights into the mechanical activation hypothesis of aGPCRs.

Piconewton Forces Mediate GAIN Domain Dissociation of the Latrophilin-3 Adhesion GPCR

“…Our data are consistent with a recent study by Fu et al in which the authors observed unfolding transitions of ∼15–20 nm at forces in the 5–10 pN range for the GAIN domains of GPR56, Lphn1, and BAI3 using a magnetic tweezers assay . Our observation of ∼13 nm steps between 4 and 5 pN therefore agrees well with the structural transitions observed in other GAIN domains, particularly when taking into account the differences between the equilibrium force measurements in this study and the force ramp experiments used by Fu et al The experiments reported by Fu et al likewise indicate that a reversible structural transition precedes dissociation for GPR56 and Lphn1 . These complementary studies thus suggest that mechanical force is likely to activate aGPCRs via a mechanism that is broadly conserved.…”