Activation of the Orphan G Protein–Coupled Receptor GPR27 by Surrogate Ligands Promotes β-Arrestin 2 Recruitment

Abstract

G protein-coupled receptors are the most important drug targets for human diseases. An important number of them remain devoid of confirmed ligands. GPR27 is one of these orphan receptors, characterized by a high level of conservation among vertebrates and a predominant expression in the central nervous system. In addition, it has recently been linked to insulin secretion. However, the absence of endogenous or surrogate ligands for GPR27 complicates the examination of its biologic function. Our aim was to validate GPR27 signaling pathways, and therefore we sought to screen a diversity-oriented synthesis library to identify GPR27-specific surrogate agonists. To select an optimal screening assay, we investigated GPR27 ligand-independent activity. Both in G protein-mediated pathways and in -arrestin 2 recruitment, no ligand-independent activity could be measured. However, we observed a recruitment of-arrestin 2 to a GPR27V chimera in the presence of membrane-anchored G protein-coupled receptor kinase-2. Therefore, we optimized a firefly luciferase complementation assay to screen against this chimeric receptor. We identified two compounds [-[4-(anilinocarbonyl)phenyl]-2,4-dichlorobenzamide (ChemBridge, San Diego, CA; ID5128535) and 2,4-dichloro--{4-[(1,3-thiazol-2-ylamino)sulfonyl]phenyl}benzamide (ChemBridge ID5217941)] sharing a -phenyl-2,4-dichlorobenzamide scaffold, which were selective for GPR27 over its closely related family members GPR85 and GPR173. The specificity of the activity was confirmed with a NanoLuc Binary Technology-arrestin 2 assay, imaging of green fluorescent protein-tagged -arrestin 2, and PathHunter-arrestin 2 assay. Interestingly, no G protein activation was detected upon activation of GPR27 by these compounds. Our study provides the first selective surrogate agonists for the orphan GPR27.

A dynamic and screening-compatible nanoluciferase-based complementation assay enables profiling of individual GPCR–G protein interactions

“…For example, systems based on the fluorescent protein of the GFP family or some ␤-galactosidases are unable to dissociate once re-formed and to accumulate in the system (38). The firefly and Gaussia luciferases seem to have a reduced propensity to form stable complexes, but the split parts have affinity for each other, and once reformed they are not very bright (39,40).…”

“…Recently, a novel protein fragment complementation based on a brighter and smaller luciferase called NanoLuc has been described (24) and was already applied in the GPCR field to detect receptor-arrestin association (24,39,41). Nano-Luc-based complementation presents several advantages compared with other RET-or complementation-based techniques.…”

GPR101 drives growth hormone hypersecretion and gigantism in mice via constitutive activation of Gs and Gq/11

“…We first established a GPR101-transfected model in Human Embryonic Kidney (HEK)-293 cells and detected a robust constitutive increase in cAMP levels using a GloSensor cAMP assay ( Fig. 4a) 22,23 . In order to firmly establish the link between cAMP production and G s , we used CRISPR/Cas9 genome editing to deplete the α subunit of the G-protein families in HEK293 cells (HEK293.ΔG s , HEK293.ΔG q/11 , and HEK293.ΔG 12/13 ) 24,25 .…”

NanoBRET: The Bright Future of Proximity-Based Assays

“…Additional recent studies by Dupuis et al (2017) and Reyes-Alcaraz et al (2018) demonstrate the utility of the NanoBiT system for monitoring GPCR-β-arrestin interactions that are important for GPCR pharmacology. NanoBiT complementation assays have also been configured to investigate other GPCR signaling processes.…”