The Orphan G Protein-coupled Receptor GPR17 Negatively Regulates Oligodendrocyte Differentiation via Gαi/o and Its Downstream Effector Molecules.

Recent studies have recognized G protein-coupled receptors as important regulators of oligodendrocyte development. GPR17, in particular, is an orphan G protein-coupled receptor that has been identified as oligodendroglial maturation inhibitor because its stimulation arrests primary mouse oligodendrocytes at a less differentiated stage. However, the intracellular signaling effectors transducing its activation remain poorly understood. Here, we use Oli-neu cells, an immortalized cell line derived from primary murine oligodendrocytes, and primary rat oligodendrocyte cultures as model systems to identify molecular targets that link cell surface GPR17 to oligodendrocyte maturation blockade. We demonstrate that stimulation of GPR17 by the small molecule agonist MDL29,951 (2-carboxy-4,6-dichloro-1H-indole-3-propionic acid) decreases myelin basic protein expression levels mainly by triggering the Gαi/o signaling pathway, which in turn leads to reduced activity of the downstream cascade adenylyl cyclase-cAMP-PKA-cAMP response element-binding protein (CREB). In addition, we show that GPR17 activation also diminishes myelin basic protein abundance by lessening stimulation of the exchange protein directly activated by cAMP (EPAC), thus uncovering a previously unrecognized role for EPAC to regulate oligodendrocyte differentiation. Together, our data establish PKA and EPAC as key downstream effectors of GPR17 that inhibit oligodendrocyte maturation. We envisage that treatments augmenting PKA and/or EPAC activity represent a beneficial approach for therapeutic enhancement of remyelination in those demyelinating diseases where GPR17 is highly expressed, such as multiple sclerosis.

A biased non-Gαi OXE-R antagonist demonstrates that Gαi protein subunit is not directly involved in neutrophil, eosinophil, and monocyte activation by 5-oxo-ETE.

Gαi-coupled chemoattractant receptors, such as the 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) receptor (OXE-R), are able to switch on Gαißγ protein-dependent and ß-arrestin-related signaling traits. However, which of these signaling pathways are truly important for the chemoattractant functions in leukocytes is not clarified yet. As we recently reported, Gue1654 is a unique Gßγ-biased OXE-R antagonist having no inhibitory activity on Gαi-related signaling, which makes Gue1654 an unprecedented tool for assessing the involvement of G protein subunits in chemoattractant receptor function. ß-arrestin2 recruitment was studied in OXE-R-overexpressing HEK293 cells using bioluminescence resonance energy transfer assays. Activation of leukocytes was assessed by flow cytometric assays and by immunofluorescence microscopy. Leukocyte capture to endothelial cells was addressed under physiological flow conditions. We found that Gue1654 blocks ß-arrestin2 recruitment in HEK293 cells overexpressing OXE-R and ERK1/2 phosphorylation in human eosinophils and neutrophils. Furthermore, Gue1654 was able to prevent several 5-oxo-ETE-triggered functional events in eosinophils and neutrophils, such as activation of CD11b/CD18 integrins, oxidative burst, actin polymerization, and interaction with endothelial cells. In addition, Gue1654 completely prevented 5-oxo-ETE-induced Ca(2+) flux and chemotaxis of human primary monocytes. All of these leukocyte responses to 5-oxo-ETE, except ERK1/2 phosphorylation and oxidative burst, were likewise prevented by pertussis toxin. Therefore, we conclude that chemoattractant receptors require Gαi subunits only as adaptors to transactivate the Gßγ heteromers, which then act responsible for cell activation. Finally, our data characterize Gue1654 as a non-Gαi-biased antagonist of OXE-R that provides a new basis for therapeutic intervention in inflammatory diseases that involve activation of eosinophils, neutrophils, and monocytes.

Decoding signaling and function of the orphan G protein-coupled receptor GPR17 with a small-molecule agonist.

Replacement of the lost myelin sheath is a therapeutic goal for treating demyelinating diseases of the central nervous system (CNS), such as multiple sclerosis (MS). The G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) GPR17, which is phylogenetically closely related to receptors of the "purinergic cluster," has emerged as a modulator of CNS myelination. However, whether GPR17-mediated signaling positively or negatively regulates this critical process is unresolved. We identified a small-molecule agonist, MDL29,951, that selectively activated GPR17 even in a complex environment of endogenous purinergic receptors in primary oligodendrocytes. MDL29,951-stimulated GPR17 engaged the entire set of intracellular adaptor proteins for GPCRs: G proteins of the Gα(i), Gα(s), and Gα(q) subfamily, as well as ß-arrestins. This was visualized as alterations in the concentrations of cyclic adenosine monophosphate and inositol phosphate, increased Ca²âº flux, phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), as well as multifeatured cell activation recorded with label-free dynamic mass redistribution and impedance biosensors. MDL29,951 inhibited the maturation of primary oligodendrocytes from heterozygous but not GPR17 knockout mice in culture, as well as in cerebellar slices from 4-day-old wild-type mice. Because GPCRs are attractive targets for therapeutic intervention, inhibiting GPR17 emerges as therapeutic strategy to relieve the oligodendrocyte maturation block and promote myelin repair in MS.

A biased ligand for OXE-R uncouples Gα and Gßγ signaling within a heterotrimer.

Differential targeting of heterotrimeric G protein versus ß-arrestin signaling are emerging concepts in G protein-coupled receptor (GPCR) research and drug discovery, and biased engagement by GPCR ligands of either ß-arrestin or G protein pathways has been disclosed. Herein we report on a new mechanism of ligand bias to titrate the signaling specificity of a cell-surface GPCR. Using a combination of biomolecular and virtual screening, we identified the small-molecule modulator Gue1654, which inhibits Gßγ but not Gα signaling triggered upon activation of Gα(i)-ßγ by the chemoattractant receptor OXE-R in both recombinant and human primary cells. Gue1654 does not interfere nonspecifically with signaling directly at or downstream of Gßγ. This hitherto unappreciated mechanism of ligand bias at a GPCR highlights both a new paradigm for functional selectivity and a potentially new strategy to develop pathway-specific therapeutics.

Applying label-free dynamic mass redistribution technology to frame signaling of G protein-coupled receptors noninvasively in living cells.

Label-free dynamic mass redistribution (DMR) is a cutting-edge assay technology that enables real-time detection of integrated cellular responses in living cells. It relies on detection of refractive index alterations on biosensor-coated microplates that originate from stimulus-induced changes in the total biomass proximal to the sensor surface. Here we describe a detailed protocol to apply DMR technology to frame functional behavior of G protein-coupled receptors that are traditionally examined with end point assays on the basis of detection of individual second messengers, such as cAMP, Ca(2+) or inositol phosphates. The method can be readily adapted across diverse cellular backgrounds (adherent or suspension), including primary human cells. Real-time recordings can be performed in 384-well microtiter plates and be completed in 2 h, or they can be extended to several hours depending on the biological question to be addressed. The entire procedure, including cell harvesting and DMR detection, takes 1-2 d.

GPR55 regulates cannabinoid 2 receptor-mediated responses in human neutrophils.

The directional migration of neutrophils towards inflammatory mediators, such as chemokines and cannabinoids, occurs via the activation of seven transmembrane G protein coupled receptors (7TM/GPCRs) and is a highly organized process. A crucial role for controlling neutrophil migration has been ascribed to the cannabinoid CB(2) receptor (CB(2)R), but additional modulatory sites distinct from CB(2)R have recently been suggested to impact CB(2)R-mediated effector functions in neutrophils. Here, we provide evidence that the recently de-orphanized 7TM/GPCR GPR55 potently modulates CB(2)R-mediated responses. We show that GPR55 is expressed in human blood neutrophils and its activation augments the migratory response towards the CB(2)R agonist 2-arachidonoylglycerol (2-AG), while inhibiting neutrophil degranulation and reactive oxygen species (ROS) production. Using HEK293 and HL60 cell lines, along with primary neutrophils, we show that GPR55 and CB(2)R interfere with each other's signaling pathways at the level of small GTPases, such as Rac2 and Cdc42. This ultimately leads to cellular polarization and efficient migration as well as abrogation of degranulation and ROS formation in neutrophils. Therefore, GPR55 limits the tissue-injuring inflammatory responses mediated by CB(2)R, while it synergizes with CB(2)R in recruiting neutrophils to sites of inflammation.

Deconvolution of complex G protein-coupled receptor signaling in live cells using dynamic mass redistribution measurements.

Label-free biosensor technology based on dynamic mass redistribution (DMR) of cellular constituents promises to translate GPCR signaling into complex optical 'fingerprints' in real time in living cells. Here we present a strategy to map cellular mechanisms that define label-free responses, and we compare DMR technology with traditional second-messenger assays that are currently the state of the art in GPCR drug discovery. The holistic nature of DMR measurements enabled us to (i) probe GPCR functionality along all four G-protein signaling pathways, something presently beyond reach of most other assay platforms; (ii) dissect complex GPCR signaling patterns even in primary human cells with unprecedented accuracy; (iii) define heterotrimeric G proteins as triggers for the complex optical fingerprints; and (iv) disclose previously undetected features of GPCR behavior. Our results suggest that DMR technology will have a substantial impact on systems biology and systems pharmacology as well as for the discovery of drugs with novel mechanisms.