The large class of adhesion G protein-coupled receptors (aGPCRs) bind extracellular matrix or neighboring cell-surface ligands to regulate organ and tissue development through an unknown activation mechanism. We examined aGPCR activation using two prototypical aGPCRs, GPR56 and GPR110. Active dissociation of the noncovalently bound GPR56 or GPR110 extracellular domains (ECDs) from the respective seven-transmembrane (7TM) domains relieved an inhibitory influence and permitted both receptors to activate defined G protein subtypes. After ECD displacement, the newly revealed short N-terminal stalk regions of the 7TM domains were found to be essential for G protein activation. Synthetic peptides comprising these stalks potently activated GPR56 or GPR110 in vitro or in cells, demonstrating that the stalks comprise a tethered agonist that was encrypted within the ECD. Establishment of an aGPCR activation mechanism provides a rational platform for the development of aGPCR synthetic modulators that could find clinical utility toward aGPCR-directed disease.

Summary Adhesion G-protein-coupled receptors (aGPCRs) play critical roles in diverse neurobiological processes including brain development, synaptogenesis, and myelination. aGPCRs have large alternatively spliced extracellular regions (ECRs) that likely mediate intercellular signaling; however, the precise roles of ECRs remain unclear. The aGPCR GPR56/ADGRG1 regulates both oligodendrocyte and cortical development. Accordingly, human GPR56 mutations cause myelination defects and brain malformations. Here, we determined the crystal structure of the GPR56 ECR, the first structure of any complete aGPCR ECR, in complex with an inverse-agonist monobody, revealing a GPCR-Autoproteolysis-Inducing domain and a previously unidentified domain that we term Pentraxin/Laminin/neurexin/sex-hormone-binding-globulin-Like (PLL). Strikingly, PLL domain deletion caused increased signaling and characterizes a GPR56 splice variant. Finally, we show that an evolutionarily conserved residue in the PLL domain is critical for oligodendrocyte development in vivo. Thus, our results suggest that the GPR56 ECR has unique and multifaceted regulatory functions, providing novel insights into aGPCR roles in neurobiology.

In the central nervous system (CNS), myelin formation and repair are regulated by oligodendrocyte (OL) lineage cells, which sense and integrate signals from their environment, including from other glial cells and the extracellular matrix (ECM). The signaling pathways that coordinate this complex communication, however, remain poorly understood. The adhesion G protein-coupled receptor ADGRG1 (also known as GPR56) is an evolutionarily conserved regulator of OL development in humans, mice, and zebrafish, although its activating ligand for OL lineage cells is unknown. Here, we report that microglia-derived transglutaminase-2 (TG2) signals to ADGRG1 on OL precursor cells (OPCs) in the presence of the ECM protein laminin and that TG2/laminin-dependent activation of ADGRG1 promotes OPC proliferation. Signaling by TG2/laminin to ADGRG1 on OPCs additionally improves remyelination in two murine models of demyelination. These findings identify a novel glia-to-glia signaling pathway that promotes myelin formation and repair, and suggest new strategies to enhance remyelination.

Circulating platelets roll along exposed collagen at vessel injury sites and respond with filipodia protrusion, shape change, and surface area expansion to facilitate platelet adhesion and plug formation. Various glycoproteins were considered to be both collagen responders and mediators of platelet adhesion, yet the signaling kinetics emanating from these receptors do not fully account for the rapid platelet cytoskeletal changes that occur in blood flow. We found the free N-terminal fragment of the adhesion G protein-coupled receptor (GPCR) GPR56 in human plasma and report that GPR56 is the platelet receptor that transduces signals from collagen and blood flow-induced shear force to activate G protein 13 signaling for platelet shape change. Gpr56−/− mice have prolonged bleeding, defective platelet plug formation, and delayed thrombotic occlusion. Human and mouse blood perfusion studies demonstrated GPR56 and shear-force dependence of platelet adhesion to immobilized collagen. Our work places GPR56 as an initial collagen responder and shear-force transducer that is essential for platelet shape change during hemostasis.

Adhesion G protein-coupled receptors (aGPCRs) have emerged as potential therapeutic targets in multiple cancers and in neurological diseases. However, there are few modulatory compounds that act on these receptors. The majority of aGPCRs are orphans and a general activation mechanism has only recently been defined: aGPCRs are activated by a tethered agonist. aGPCRs constitutively cleave themselves during biosynthesis to generated two-part receptors comprised of an extracellular domain (ECD) and a 7-transmembrane spanning domain (7TM). ECD dissociation reveals the tethered agonist, initiating G protein signaling. Synthetic peptides that mimic the tethered agonist region can activate aGPCRs. We hypothesized that small molecules could act similarly as peptide agonists. High throughput screening of the 2000 compound Spectrum Collection library using the Serum Response Element luciferase gene reporter assay revealed two related classes of small molecules that could activate the aGPCR GPR56 / ADGRG1. The most potent compound identified was 3-a-acetoxydihydrodeoxygedunin, or 3-a-DOG. 3-a-DOG activated engineered, low-activity GPR56 7TM in independent biochemical and cell-based assays with an EC 50 of ~5 µM. The compound also activated a subset of aGPCRs but not two Class A GPCRs tested. The mode of 3-a-DOG-mediated receptor activation is as a partial agonist. 3-a-DOG activated GPR56 less efficaciously than peptide agonist and could antagonize both the peptide agonist and the endogenous tethered agonist, which are pharmacological hallmarks of partial agonists. Taken together, we have uncovered a novel group of aGPCR partial agonists that will serve as invaluable resources for understanding this unique class receptors.