<i>In vitro</i> profiling of orphan G protein coupled receptor (GPCR) constitutive activity

Watkins, Lyndsay R.; Orlandi, Cesare

doi:10.1111/bph.15468

Cited by 33 publications

(26 citation statements)

References 57 publications

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“…Many of our high-basal and agonist-dependent findings are in agreement with previous studies, such as high constitutive activity at GPR182-Tango receptor (Fig. 6I) 35,36 . Moreover, Tango-Trio was able to detect novel activity at GPCRs that could not be validated in PRESTO-Tango 13 , including BAM-22 at MRGRPX2-Tango (Fig.…”

Section: Validation Of Gpcr Internalization β-Arrestin-1/2 Coupling A...supporting

confidence: 93%

Profiling of basal and ligand-dependent GPCR activities by means of a polyvalent cell-based high-throughput platform

Giguère

Zeghal

Laroche

et al. 2023

Preprint

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Representing the most attractive and successful druggable receptors of the proteome, GPCRs regulate a myriad of physiological and pathophysiological functions. Although over half of present pharmaceuticals target GPCRs, the advancement of drug discovery is hampered by a lack of adequate screening tools, the majority of which are limited to probing agonist-induced G-protein and β-arrestin-2-mediated events as a measure of receptor activation. Here, we develop Tango-Trio, a comprehensive cell-based high-throughput platform capable of the parallelized profiling of both basal and agonist-dependent GPCR activities. We capture the functional diversity of GPCRs, reporting β-arrestin-1/2 couplings, selectivities, and receptor internalization signatures across the GPCRome. Moreover, we present the first construction of dose-dependent basal activation curves at approximately 200 receptors, including over 50 orphans. Overall, Tango-Trio’s robustness is well-suited for the functional characterization and screening of GPCRs, especially for parallel interrogation, and is a valuable addition to the pharmacological toolbox.

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Section: Validation Of Gpcr Internalization β-Arrestin-1/2 Coupling A...supporting

confidence: 93%

Profiling of basal and ligand-dependent GPCR activities by means of a polyvalent cell-based high-throughput platform

Giguère

Zeghal

Laroche

et al. 2023

Preprint

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“…Activation of the G protein by GPR158 remains unclear: although OCN binds to a complex containing GPR158 and Gαq and regulates the IP3 production, no functional assay using OCN as an agonist has been reported 2 . By contrast, GPR158 exhibits constitutive activity for Gi/o proteins but not for Gq 9 . The noncanonical signaling for GPR158 is more clearly established: GPR158 localizes RGS7–Gβ5 and the Gαi/o protein activated by other GPCRs, and allosterically promotes GTPase activity of Gαi/o, which ultimately reduces the activity of adenylate cyclase and controls other signaling pathways 7 , 8 , 10 , 11 .…”

Section: Introductionmentioning

confidence: 89%

Structure of the class C orphan GPCR GPR158 in complex with RGS7-Gβ5

et al. 2021

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GPR158, a class C orphan GPCR, functions in cognition, stress-induced mood control, and synaptic development. Among class C GPCRs, GPR158 is unique as it lacks a Venus flytrap-fold ligand-binding domain and terminates Gαi/o protein signaling through the RGS7-Gβ5 heterodimer. Here, we report the cryo-EM structures of GPR158 alone and in complex with one or two RGS7-Gβ5 heterodimers. GPR158 dimerizes through Per-Arnt-Sim-fold extracellular and transmembrane (TM) domains connected by an epidermal growth factor-like linker. The TM domain (TMD) reflects both inactive and active states of other class C GPCRs: a compact intracellular TMD, conformations of the two intracellular loops (ICLs) and the TMD interface formed by TM4/5. The ICL2, ICL3, TM3, and first helix of the cytoplasmic coiled-coil provide a platform for the DHEX domain of one RGS7 and the second helix recruits another RGS7. The unique features of the RGS7-binding site underlie the selectivity of GPR158 for RGS7.

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“…Cloning of constructs for the mammalian expression of GPRC5A, GPRC5B, GPRC5C, GPRC5D, was previously described ( Watkins and Orlandi, 2021 ). HEK293T/17 cells were used for transfection and western blotting analysis.…”

Section: Methodsmentioning

confidence: 99%

Comprehensive Spatial Profile of the Orphan G Protein Coupled Receptor GPRC5B Expression in Mouse Brain

Franchini

Orlandi

2022

Front. Neurosci.

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Orphan G Protein Coupled Receptors (GPCRs) are GPCRs whose endogenous ligands are unknown or still debated. Due to the lack of pharmacological modulators, the physiological function of orphan GPCRs is understudied. However, relevant physiological roles associated with orphan GPCRs have been revealed by analysis of animal models and genome wide association studies illuminating an untapped potential for drug discovery. G Protein Coupled Receptor class C Group 5 Member B (GPRC5B) is among the most expressed GPCRs in the central nervous system. Thus, the expression profiling of GPRC5B is an essential step toward understanding GPRC5B function in health and disease. In this study, we generated new GPRC5B polyclonal antibodies and investigated the expression levels of GPRC5B across different organs and brain regions. We identified high levels of GPRC5B glycosylation both in transfected cells and in mouse brain. Moreover, in situ hybridization imaging analysis indicated that Gprc5b was expressed at the highest level in olfactory bulb, hippocampus, cerebellum, and pons. To dissect expression within various neuronal populations, we conducted a comprehensive spatial profiling of Gprc5b across excitatory and inhibitory neuronal types in medial prefrontal cortex, motor cortex, hippocampal regions, hypothalamus, and cerebellum. Overall, we discovered that GABAergic neurons displayed higher Gprc5b expression levels than glutamatergic neurons in most of the analyzed regions with the important exception of the hippocampal dentate gyrus. Overall, the expression analysis of GPRC5B in mouse brain will guide functional studies ultimately positioning GPRC5B in pathophysiological mechanisms and drug discovery.

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In vitro profiling of orphan G protein coupled receptor (GPCR) constitutive activity

Cited by 33 publications

References 57 publications

Profiling of basal and ligand-dependent GPCR activities by means of a polyvalent cell-based high-throughput platform

Profiling of basal and ligand-dependent GPCR activities by means of a polyvalent cell-based high-throughput platform

Structure of the class C orphan GPCR GPR158 in complex with RGS7-Gβ5

Comprehensive Spatial Profile of the Orphan G Protein Coupled Receptor GPRC5B Expression in Mouse Brain

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