Activation of GPR97/ADGRG3 by its tethered agonist, but not by beclomethasone, induces neutrophil polarization and migration

Most adhesion G protein-coupled receptors (AGPCRs) are activated by the intramolecular binding of a tethered agonist, which is exposed by shear force-induced dissociation of the N- and C-terminal fragments of AGPCRs. The decrypted tethered agonist binds to its orthosteric site in the C-terminal fragment to stabilize the active state of the AGPCR. Corticosteroids have been proposed to be orthosteric agonists for GPR97/ADGRG3. Here, we showed that GPR97/ADGRG3 is activated by the canonical tethered agonist mechanism. In cell-based luciferase reporter assays and receptor/G protein reconstitution assays, GPR97/ADGRG3 was stimulated by agonist peptidomimetics and by 3-acetoxydihydrodeoxygeduin (3-α-DOG), a partial agonist of the ADGRG subfamily, but not by corticosteroids. We showed that GPR97 was a promiscuous AGPCR that coupled to G₁₃, G_s, and G_i but not to G_q. GPR97 is abundant in neutrophils, which undergo cell shape changes and polarization and migrate upon activation. GPR97 activation by tethered agonist peptidomimetics or 3-α-DOG robustly stimulated cAMP production, polarization, and chemotaxis in human and mouse neutrophils. These effects in mouse neutrophils required GPR97 and were not mimicked by the corticosteroid beclomethasone in either cell type. Together, our results demonstrate that GPR97/ADGRG3 uses a tethered agonist mechanism to activate G protein signaling and induce neutrophil polarization and migration.