Novel Screening Assay for the Selective Detection of G-Protein-Coupled Receptor Heteromer Signaling

Rijn, Richard M. van; Harvey, Jessica H.; Brissett, Daniela I.; DeFriel, Julia N.; Whistler, Jennifer L.

doi:10.1124/jpet.112.198655

Cited by 17 publications

(13 citation statements)

References 45 publications

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“…Twenty-five µl calcium sensitive Fluorescent Imaging Plate Reader (FLIPR) Calcium 6 assay dye (#R8190, Molecular Devices, CA, USA) was added to each well of a 384-well plate containing HEK293 cells transiently expressing mTRPA1, zTRPA1a, and zTRPA1b, respectively. The cells were incubated for an hour prior to the recording of intracellular calcium levels in a FlexStation3 Multi-Mode Microplate Reader (#R8190, Molecular Devices) as previously described 40 . All compounds were diluted in calcium buffer made with 1x HBSS (#14025-092, Thermo Fisher), 20 mM HEPES (#15630-080, Thermo Fisher), and 2.5 mM Probenecid (#P8761, Sigma-Aldrich).…”

Section: Methodsmentioning

confidence: 99%

A critical evaluation of TRPA1-mediated locomotor behavior in zebrafish as a screening tool for novel anti-nociceptive drug discovery

Ganzen

Coskun³

et al. 2019

Sci Rep

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Current medications inadequately treat the symptoms of chronic pain experienced by over 50 million people in the United States, and may come with substantial adverse effects signifying the need to find novel treatments. One novel therapeutic target is the Transient Receptor Potential A1 channel (TRPA1), an ion channel that mediates nociception through calcium influx of sensory neurons. Drug discovery still relies heavily on animal models, including zebrafish, a species in which TRPA1 activation produces hyperlocomotion. Here, we investigated if this hyperlocomotion follows zebrafish TRPA1 pharmacology and evaluated the strengths and limitations of using TRPA1-mediated hyperlocomotion as potential preclinical screening tool for drug discovery. To support face validity of the model, we pharmacologically characterized mouse and zebrafish TRPA1 in transfected HEK293 cells using calcium assays as well as in vivo. TRPA1 agonists and antagonists respectively activated or blocked TRPA1 activity in HEK293 cells, mice, and zebrafish in a dose-dependent manner. However, our results revealed complexities including partial agonist activity of TRPA1 antagonists, bidirectional locomotor activity, receptor desensitization, and off-target effects. We propose that TRPA1-mediated hyperlocomotion in zebrafish larvae has the potential to be used as in vivo screening tool for novel anti-nociceptive drugs but requires careful evaluation of the TRPA1 pharmacology.

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Section: Methodsmentioning

confidence: 99%

A critical evaluation of TRPA1-mediated locomotor behavior in zebrafish as a screening tool for novel anti-nociceptive drug discovery

Ganzen

Coskun³

et al. 2019

Sci Rep

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“…Cells were then loaded with 25 μl of the FLIPR Ca 2+ dye (Molecular Devices, Sunnyvale, CA, USA) and incubated for 1 h at 37 °C. Agonists were added in a 20 μl volume and calcium mobilization was measured in real-time for 2 min in a Flexstation 3 apparatus (Molecular Devices, Sunnyvale, CA, USA) and analyzed as described in detail in van Rijn et al . (2013).…”

Section: Methodsmentioning

confidence: 99%

Functional histamine H 3 and adenosine A 2A receptor heteromers in recombinant cells and rat striatum

Márquez-Gómez

Robins

Gutiérrez-Rodelo

et al. 2018

Pharmacological Research

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In the striatum, histamine H receptors (HRs) are co-expressed with adenosine A receptors (ARs) in the cortico-striatal glutamatergic afferents and the GABAergic medium-sized spiny neurons that originate the indirect pathway of the basal ganglia. This location allows HRs and ARs to regulate the striatal GABAergic and glutamatergic transmission. However, whether these receptors can physically interact has not yet been assessed. To test this hypothesis, a heteromer-selective in vitro assay was used to detect functional complementation between a chimeric AR-Gα and wild-type HRs in transfected HEK-293T cells. HR activation with the agonist RAMH resulted in Ca mobilization (pEC 7.31 ± 0.23; maximal stimulation, Emax 449 ± 25% of basal) indicative of receptor heterodimerization. Functional HR-AR heteromers were confirmed by co-immunoprecipitation and observations of differential cAMP signaling when both receptors were co-expressed in the same cells. In membranes from rat striatal synaptosomes, HR activation decreased AR affinity for the agonist CGS-21680 (pKi values 8.10 ± 0.04 and 7.70 ± 0.04). Moreover, HRs and ARs co-immunoprecipitated in protein extracts from striatal synaptosomes. These results support the existence of a HR-AR heteromer with possible physiological implications for the modulation of the intra-striatal transmission.

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“…Bioluminescence resonance energy transfer-based approaches Watts et al, 2013), modified GPCR-G protein fusion systems (Han et al, 2009;van Rijn et al, 2013), or bivalent ligands (Waldhoer et al, 2005;Ferre et al, 2014) have all been used to assign pharmacology to defined heterodimers. Such investigations can depend on ligand selectivity, cross-talk between signaling pathways and the allosteric influence of coexpressed effector proteins, and the potential for receptor dimers to be transient in nature (Dorsch et al, 2009;Fonseca and Lambert, 2009).…”

Section: Discussionmentioning

confidence: 99%

A G Protein–Coupled Receptor Dimer Imaging Assay Reveals Selectively Modified Pharmacology of Neuropeptide Y Y1/Y5 Receptor Heterodimers

2015

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The ability of G protein-coupled receptors (GPCRs) to form dimers, and particularly heterodimers, offers potential for targeted therapeutics with improved selectivity. However, studying dimer pharmacology is challenging, because of signaling cross-talk or because dimerization may often be transient in nature. Here we develop a system to isolate the pharmacology of precisely defined GPCR dimers, trapped by bimolecular fluorescence complementation (BiFC). Specific effects of agonist activation on such dimers are quantified using automated imaging and analysis of their internalization, controlled for by simultaneous assessment of endocytosis of one coexpressed protomer population. We applied this BiFC system to study example neuropeptide Y (NPY) Y1 receptor dimers. Incorporation of binding-site or phosphorylation-site mutations into just one protomer of a Y1/Y1 BiFC homodimer had no impact on efficient NPY-stimulated endocytosis, demonstrating that single-site agonist occupancy, and one phosphorylated monomer within this dimer, was sufficient. For two Y1 receptor heterodimer combinations (with the Y4 receptor or b2-adrenoceptor), agonist and antagonist pharmacology was explained by independent actions on the respective orthosteric binding sites. However, Y1/Y5 receptor BiFC dimers, compared with the constituent subtypes, were characterized by reduced potency and efficacy of Y5-selective peptide agonists, the inactivity of Y1-selective antagonists, and a change from surmountable to nonsurmountable antagonism for three unrelated Y5 antagonists. Thus, allosteric interactions between Y1 and Y5 receptors modify the pharmacology of the heterodimer, with implications for potential antiobesity agents that target centrally coexpressed Y1 and Y5 receptors to suppress appetite.

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Novel Screening Assay for the Selective Detection of G-Protein-Coupled Receptor Heteromer Signaling

Cited by 17 publications

References 45 publications

A critical evaluation of TRPA1-mediated locomotor behavior in zebrafish as a screening tool for novel anti-nociceptive drug discovery

A critical evaluation of TRPA1-mediated locomotor behavior in zebrafish as a screening tool for novel anti-nociceptive drug discovery

Functional histamine H 3 and adenosine A 2A receptor heteromers in recombinant cells and rat striatum

A G Protein–Coupled Receptor Dimer Imaging Assay Reveals Selectively Modified Pharmacology of Neuropeptide Y Y1/Y5 Receptor Heterodimers

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