An enduring challenge in the adhesion G protein-coupled receptor (AGPCR) field is achieving a comprehensive understanding of the activation mechanisms governing this 33-member receptor class. Structural insights, with active-state structures resolved for multiple AGPCRs, have illuminated how these receptors respond to endogenous full tethered agonists. AGPCRs are uniquely self-activated through a tethered peptide agonist (TA), which shifts from an encrypted, concealed state within the GPCR autoproteolytic inducing (GAIN) domain to a decrypted TA that rapidly engages the orthosteric binding pocket typical of GPCRs. We trace the key milestones that led to the discovery of the TA activation mechanism. Steroid-bound AGPCRs structures continue to be resolved promoting controversy in the field as steroids are being suggested as full agonists of GPR97, our evaluation of their activation potential revealed minimal to no activation, whereas 3-a-DOG exhibited partial agonism in both membrane signaling and cell-based luciferase assays. Our analysis of the available structures further supports our hypothesis.
The G Family AGPCR members GPR97, GPR114, and GPR56 are gene duplications of each other; therefore, opening the question if all 3 of these AGPCRs are characteristically similar. GPR56 is a hallmark AGPCR with a multitude of studies showing cleavage-dependent tethered agonism. We sought to show that GPR97 and GPR114 are also cleavage-dependent AGPCRs activated by their TA as this was never shown before for GPR97 and GPR114 was purported to be a cleavage-deficient AGPCR that activated through alternative modes. Using immunoblotting and biochemical assays, we demonstrate that GPR97 and GPR114 undergo self-cleavage, which is essential for TA-mediated activation. We used GPCR/G protein reconstitution assays and whole cell signaling assays to determine and validate G protein coupling profiles for these two receptors. GPR97 showed coupling to Gs, G13, and Gi but not Gq whereas, GPR114 solely showed coupling to Gs. In the case for GPR114, we used mutagenesis studies to identify key features within the GAINA subdomain that influence self-cleavage efficiency.
We explore the potential role of extracellular shear forces in initiating TA decryption in physiological contexts. GPR56 was shown to have a physiological role on platelets. Therefore, we wanted to examine what GPR97 and GPR114 roles are in other circulating blood cells where they are expressed, neutrophils and eosinophils, respectively. We found that GPR114 undergoes efficient endogenous self-cleavage in an eosinophilic-like cell line (EoL-1 cells). Application of GPR114 TA peptidomimetics to EoL-1 cells stimulated cAMP production, suggesting a functional role in eosinophil signaling. We also found that GPR97 activation by TA peptidomimetics and 3-a-DOG induced actin polymerization and migration in neutrophils.
Together, we have confirmed the tethered agonist activation mechanism for GPR97 and GPR114 while providing insights into the cellular roles of GPR97 and GPR114 in white blood cell processes such as migration, chemotaxis, and potentially degranulation. Ultimately providing the groundwork for the future of AGPCR physiology and pharmacology research.