Abstract
Adhesion G protein-coupled receptors (aGPCRs) represent a distinct family of GPCRs that regulate several developmental and physiological processes. Most aGPCRs undergo GPCR autoproteolysis-inducing domain-mediated protein cleavage, which produces a cryptic tethered agonist (termed Stachel (stinger)), and cleavage-dependent and -independent aGPCR signaling mechanisms have been described. aGPCR G1 (ADGRG1 or G protein-coupled receptor 56 (GPR56)) has pleiotropic functions in the development of multiple organ systems, which has broad implications for human diseases. To date, two natural GPR56 ligands, collagen III and tissue transglutaminase (TG2), and one small-molecule agonist, 3-α-acetoxydihydrodeoxygedunin (3-α-DOG), have been identified, in addition to a synthetic peptide, P19, that contains seven amino acids of the native Stachel sequence. However, the mechanisms by which these natural and small-molecule agonists signal through GPR56 remain unknown. Here we engineered a noncleavable receptor variant that retains signaling competence via the P19 peptide. We demonstrate that both natural and small-molecule agonists can activate only cleaved GPR56. Interestingly, TG2 required both receptor cleavage and the presence of a matrix protein, laminin, to activate GPR56, whereas collagen III and 3-α-DOG signaled without any cofactors. On the other hand, both TG2/laminin and collagen III activate the receptor by dissociating the N-terminal fragment from its C-terminal fragment, enabling activation by the Stachel sequence, whereas P19 and 3-α-DOG initiate downstream signaling without disengaging the N-terminal fragment from its C-terminal fragment. These findings deepen our understanding of how GPR56 signals via natural ligands, and a small-molecule agonist may be broadly applicable to other aGPCR family members.