GPR56/ADGRG1 is a platelet collagen-responsive GPCR and hemostatic sensor of shear force.

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.

G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.

The adhesion G-protein-coupled receptor (GPCR) latrophilin 3 (ADGRL3) has been associated with increased risk of attention deficit hyperactivity disorder (ADHD) and substance use in human genetic studies. Knockdown in multiple species leads to hyperlocomotion and altered dopamine signaling. Thus, ADGRL3 is a potential target for treatment of neuropsychiatric disorders that involve dopamine dysfunction, but its basic signaling properties are poorly understood. Identification of adhesion GPCR signaling partners has been limited by a lack of tools to acutely activate these receptors in living cells. Here, we design a novel acute activation strategy to characterize ADGRL3 signaling by engineering a receptor construct in which we could trigger acute activation enzymatically. Using this assay, we found that ADGRL3 signals through G12/G13 and Gq, with G12/13 the most robustly activated. Gα12/13 is a new player in ADGRL3 biology, opening up unexplored roles for ADGRL3 in the brain. Our methodological advancements should be broadly useful in adhesion GPCR research.

Publisher Correction: G12/13 is activated by acute tethered agonist exposure in the adhesion GPCR ADGRL3.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The orphan receptor GPR139 signals via Gq/11 to oppose opioid effects.

The interplay between G protein-coupled receptors (GPCRs) is critical for controlling neuronal activity that shapes neuromodulatory outcomes. Recent evidence indicates that the orphan receptor GPR139 influences opioid modulation of key brain circuits by opposing the actions of the µ-opioid receptor (MOR). However, the function of GPR139 and its signaling mechanisms are poorly understood. In this study, we report that GPR139 activates multiple heterotrimeric G proteins, including members of the Gq/11 and Gi/o families. Using a panel of reporter assays in reconstituted HEK293T/17 cells, we found that GPR139 functions via the Gq/11 pathway and thereby distinctly regulates cellular effector systems, including stimulation of cAMP production and inhibition of G protein inward rectifying potassium (GIRK) channels. Electrophysiological recordings from medial habenular neurons revealed that GPR139 signaling via Gq/11 is necessary and sufficient for counteracting MOR-mediated inhibition of neuronal firing. These results uncover a mechanistic interplay between GPCRs involved in controlling opioidergic neuromodulation in the brain.

Gedunin- and Khivorin-Derivatives Are Small-Molecule Partial Agonists for Adhesion G Protein-Coupled Receptors GPR56/ADGRG1 and GPR114/ADGRG5.

Adhesion G protein-coupled receptors (aGPCRs) have emerged as potential therapeutic targets in multiple cancers and in neurologic 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 comprising 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 in the same way 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-α-acetoxydihydrodeoxygedunin, or 3-α-DOG. 3-α-DOG activated engineered, low-activity GPR56 7TM in independent biochemical and cell-based assays with an EC50 of ∼5 µM. The compound also activated a subset of aGPCRs but not two class A GPCRs tested. The mode of 3-α-DOG-mediated receptor activation is that of partial agonist. 3-α-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.

Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist.

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.

Dihydromunduletone Is a Small-Molecule Selective Adhesion G Protein-Coupled Receptor Antagonist.

Adhesion G protein-coupled receptors (aGPCRs) have emerging roles in development and tissue maintenance and is the most prevalent GPCR subclass mutated in human cancers, but to date, no drugs have been developed to target them in any disease. aGPCR extracellular domains contain a conserved subdomain that mediates self-cleavage proximal to the start of the 7-transmembrane domain (7TM). The two receptor protomers, extracellular domain and amino terminal fragment (NTF), and the 7TM or C-terminal fragment remain noncovalently bound at the plasma membrane in a low-activity state. We recently demonstrated that NTF dissociation liberates the 7TM N-terminal stalk, which acts as a tethered-peptide agonist permitting receptor-dependent heterotrimeric G protein activation. In many cases, natural aGPCR ligands are extracellular matrix proteins that dissociate the NTF to reveal the tethered agonist. Given the perceived difficulty in modifying extracellular matrix proteins to create aGPCR probes, we developed a serum response element (SRE)-luciferase-based screening approach to identify GPR56/ADGRG1 small-molecule inhibitors. A 2000-compound library comprising known drugs and natural products was screened for GPR56-dependent SRE activation inhibitors that did not inhibit constitutively active Gα13-dependent SRE activation. Dihydromunduletone (DHM), a rotenoid derivative, was validated using cell-free aGPCR/heterotrimeric G protein guanosine 5'-3-O-(thio)triphosphate binding reconstitution assays. DHM inhibited GPR56 and GPR114/ADGRG5, which have similar tethered agonists, but not the aGPCR GPR110/ADGRF1, M3 muscarinic acetylcholine, or ß2 adrenergic GPCRs. DHM inhibited tethered peptide agonist-stimulated and synthetic peptide agonist-stimulated GPR56 but did not inhibit basal activity, demonstrating that it antagonizes the peptide agonist. DHM is a novel aGPCR antagonist and potentially useful chemical probe that may be developed as a future aGPCR therapeutic.

The role of orphan receptor GPR139 in neuropsychiatric behavior.

Orphan G protein Coupled Receptors (GPCRs) present attractive targets both for understanding neuropsychiatric diseases and for development of novel therapeutics. GPR139 is an orphan GPCR expressed in select brain circuits involved in controlling movement, motivation and reward. It has been linked to the opioid and dopamine neuromodulatory systems; however, its role in animal behavior and neuropsychiatric processes is poorly understood. Here we present a comprehensive behavioral characterization of a mouse model with a GPR139 null mutation. We show that loss of GPR139 in mice results in delayed onset hyperactivity and prominent neuropsychiatric manifestations including elevated stereotypy, increased anxiety-related traits, delayed acquisition of operant responsiveness, disruption of cued fear conditioning and social interaction deficits. Furthermore, mice lacking GPR139 exhibited complete loss of pre-pulse inhibition and developed spontaneous 'hallucinogenic' head-twitches, altogether suggesting schizophrenia-like symptomatology. Remarkably, a number of these behavioral deficits could be rescued by the administration of µ-opioid and D2 dopamine receptor (D2R) antagonists: naltrexone and haloperidol, respectively, suggesting that loss of neuropsychiatric manifestations in mice lacking GPR139 are driven by opioidergic and dopaminergic hyper-functionality. The inhibitory influence of GPR139 on D2R signaling was confirmed in cell-based functional assays. These observations define the role of GPR139 in controlling behavior and implicate in vivo actions of this receptor in the neuropsychiatric process with schizophrenia-like pathology.

Ptchd1 mediates opioid tolerance via cholesterol-dependent effects on µ-opioid receptor trafficking.

Repeated exposure to opioids causes tolerance, which limits their analgesic utility and contributes to overdose and abuse liability. However, the molecular mechanisms underpinning tolerance are not well understood. Here, we used a forward genetic screen in Caenorhabditis elegans for unbiased identification of genes regulating opioid tolerance which revealed a role for PTR-25/Ptchd1. We found that PTR-25/Ptchd1 controls µ-opioid receptor trafficking and that these effects were mediated by the ability of PTR-25/Ptchd1 to control membrane cholesterol content. Electrophysiological studies showed that loss of Ptchd1 in mice reduced opioid-induced desensitization of neurons in several brain regions and the peripheral nervous system. Mice and C. elegans lacking Ptchd1/PTR-25 display similarly augmented responses to opioids. Ptchd1 knockout mice fail to develop analgesic tolerance and have greatly diminished somatic withdrawal. Thus, we propose that Ptchd1 plays an evolutionarily conserved role in protecting the µ-opioid receptor against overstimulation.

Identification of Potential Modulators of the RGS7/Gß5/R7BP Complex.

Regulators of G protein signaling (RGS) proteins serve as critical regulatory nodes to limit the lifetime and extent of signaling via G protein-coupled receptors (GPCRs). Previously, approaches to pharmacologically inhibit RGS activity have mostly focused on the inhibition of GTPase activity by interrupting the interaction of RGS proteins with the G proteins they regulate. However, several RGS proteins are also regulated by association with binding partners. A notable example is the mammalian RGS7 protein, which has prominent roles in metabolic control, vision, reward, and actions of opioid analgesics. In vivo, RGS7 exists in complex with the binding partners type 5 G protein ß subunit (Gß5) and R7 binding protein (R7BP), which control its stability and activity, respectively. Targeting the whole RGS7/Gß5/R7BP protein complex affords the opportunity to allosterically tune opioid receptor signaling following opioid engagement while potentially bypassing undesirable side effects. Hence, we implemented a novel strategy to pharmacologically target the interaction between RGS7/Gß5 and R7BP. To do so, we searched for protein complex inhibitors using a time-resolved fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) assay that measures compound-mediated alterations in the FRET signal between RGS7/Gß5 and R7BP. We performed two HTS campaigns, each screening ~100,000 compounds from the Scripps Drug Discovery Library (SDDL). Each screen yielded more than 100 inhibitors, which will be described herein.

Genetic behavioral screen identifies an orphan anti-opioid system.

Opioids target the µ-opioid receptor (MOR) to produce unrivaled pain management, but their addictive properties can lead to severe abuse. We developed a whole-animal behavioral platform for unbiased discovery of genes influencing opioid responsiveness. Using forward genetics in Caenorhabditis elegans, we identified a conserved orphan receptor, GPR139, with anti-opioid activity. GPR139 is coexpressed with MOR in opioid-sensitive brain circuits, binds to MOR, and inhibits signaling to heterotrimeric guanine nucleotide-binding proteins (G proteins). Deletion of GPR139 in mice enhanced opioid-induced inhibition of neuronal firing to modulate morphine-induced analgesia, reward, and withdrawal. Thus, GPR139 could be a useful target for increasing opioid safety. These results also demonstrate the potential of C. elegans as a scalable platform for genetic discovery of G protein-coupled receptor signaling principles.

Dual phosphorylation of Ric-8A enhances its ability to mediate G protein α subunit folding and to stimulate guanine nucleotide exchange.

Resistance to inhibitors of cholinesterase-8A (Ric-8A) and Ric-8B are essential biosynthetic chaperones for heterotrimeric G protein α subunits. We provide evidence for the direct regulation of Ric-8A cellular activity by dual phosphorylation. Using proteomics, Western blotting, and mutational analyses, we determined that Ric-8A was constitutively phosphorylated at five serines and threonines by the protein kinase CK2. Phosphorylation of Ser435 and Thr440 in rat Ric-8A (corresponding to Ser436 and Thr441 in human Ric-8A) was required for high-affinity binding to Gα subunits, efficient stimulation of Gα subunit guanine nucleotide exchange, and mediation of Gα subunit folding. The CK2 consensus sites that contain Ser435 and Thr440 are conserved in Ric-8 homologs from worms to mammals. We found that the homologous residues in mouse Ric-8B, Ser468 and Ser473, were also phosphorylated. Mutation of the genomic copy of ric-8 in Caenorhabditis elegans to encode alanine in the homologous sites resulted in characteristic ric-8 reduction-of-function phenotypes that are associated with defective Gq and Gs signaling, including reduced locomotion and defective egg laying. The C. elegans ric-8 phosphorylation site mutant phenotypes were partially rescued by chemical stimulation of Gq signaling. These results indicate that dual phosphorylation represents a critical form of conserved Ric-8 regulation and demonstrate that Ric-8 proteins are needed for effective Gα signaling. The position of the CK2-phosphorylated sites within a structural model of Ric-8A reveals that these sites contribute to a key acidic and negatively charged surface that may be important for its interactions with Gα subunits.

Microglial transglutaminase-2 drives myelination and myelin repair via GPR56/ADGRG1 in oligodendrocyte precursor cells.

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.

Structural Basis for Regulation of GPR56/ADGRG1 by Its Alternatively Spliced Extracellular Domains.

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.