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.
Highlights
Adhesion G protein– coupled receptors represent a distinct family of GPCRs that regulate several developmental and physiological processes
Western blot analysis revealed that GPR56H381S completely lacks the cleaved N-terminal fragment (NTF), whereas ϳ 56% of the GPR56L382A is cleaved (Fig. 1, F and G), showing that the GPR56H381S receptor is defective in undergoing GPCR autoproteolysis-inducing (GAIN)
The evidence to date indicates Adhesion G protein– coupled receptors (aGPCRs) signaling modes that are either GAIN domain–mediated receptor cleavage– dependent or cleavage-independent [1, 3, 6, 7, 9, 11,12,13]
Summary
To investigate the requirement of GAIN domain–mediated autoproteolysis for GPR56 signaling, we first generated GPR56 mutants that abolish receptor cleavage but retain cell surface expression. Only P19 could elicit signaling from the GPR56H381S mutant receptor (Fig. 2) To further confirm this observation, we performed GTP-RhoA pulldown assays to measure levels of activated RhoA upon ligand stimulation. Treatment with 3-␣-DOG, P19, and collagen III resulted in robust RhoA activation (Fig. 2, B and C) This result indicated that the GPR56H381S mutant receptor retained signaling competence but did not respond to natural or smallmolecule agonist stimulation. Our findings support that 3-␣DOG is a partial orthosteric agonist that requires the presence of a partial Stachel sequence to activate the receptor
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