Monitoring G Protein Activation in Cells with BRET

Masuho, Ikuo; Martemyanov, Kirill A.; Lambert, Nevin A.

doi:10.1007/978-1-4939-2914-6_8

Cited by 76 publications

(77 citation statements)

References 9 publications

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“…The prevalence of disruptive GNAL mutations in this cohort of dystonia patients was 1.09%, which is consistent with those recently reported between 0.5% and 2.6% among individuals of European and Asian ancestry [8–11]. In Brazilian patients, idiopathic dystonia associated to GNAL mutations is less frequent than dystonia associated to THAP1 (8.8%) or TOR1A (2.6%) mutations [3].…”

Section: Discussionsupporting

confidence: 90%

“…Functional characterization was performed using the improved bioluminescence resonance energy transfer (BRET) assay [11] similar in design and concept to the assay previously used to evaluate the effect of mutations in Gα olf [4]. Briefly, constructs encoding dopamine D1 receptor, Gα olf , Venus155–239–Gβ 1 , Venus1–155–Gγ 2 , masGRK3ct-Nluc, PTX-S1 and Flag-tagged Ric-8B were transfected into HEK293/17 cells at a 1:6:1:1:1:1:1 ratio, with 7.5 μg of total DNA delivered per 3.5 × 10 6 cells.…”

Section: Methodsmentioning

confidence: 99%

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Screening of GNAL variants in Brazilian patients with isolated dystonia reveals a novel mutation with partial loss of function

et al. 2016

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GNAL was identified as a cause of dystonia in patients from North America, Europe and Asia. In this study, we aimed to investigate the prevalence of GNAL variants in Brazilian patients with dystonia. Ninety-one patients with isolated idiopathic dystonia, negative for THAP1 and TOR1A mutations, were screened for GNAL variants by Sanger sequencing. Functional characterization of the Gαolf protein variant was performed using the bioluminescence resonance energy transfer assay. A novel heterozygous nonsynonymous variant (p.F133L) was identified in a patient with cervical and laryngeal dystonia since the third decade of life, with no family history. This variant was not identified in healthy Brazilian controls and was not described in 63000 exomas of the ExAC database. The F133L mutant exhibited significantly elevated levels of basal BRET and severely diminished amplitude of response elicited by dopamine, that both indicate su bstantial functional impairment of Gαolf in transducing receptor signals, which could be involved in dystonia pathophysiology. GNAL mutations are not a common cause of dystonia in the Brazilian population and have a lower prevalence than THAP1 and TOR1A mutations. We present a novel variant that results in partial Gαolf loss of function.

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Section: Discussionsupporting

confidence: 90%

Section: Methodsmentioning

confidence: 99%

Screening of GNAL variants in Brazilian patients with isolated dystonia reveals a novel mutation with partial loss of function

et al. 2016

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“…The largest agonist-induced ΔBRET ratio was plotted as the maximum BRET amplitude. Cellular measurements of BRET between Venus-Gβγ and masGRK3ct-Nluc were performed (Masuho et al, 2015a; Masuho et al, 2015b) with minor modifications to monitor G protein activity in real-time with BRET sensors.…”

Section: Resultsmentioning

confidence: 99%

Dopamine Receptor DAMB Signals via Gq to Mediate Forgetting in Drosophila

et al. 2017

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Prior studies have shown that aversive olfactory memory is acquired by dopamine acting on a specific receptor, dDA1, expressed by mushroom body neurons. Active forgetting is mediated by dopamine acting on another receptor, Damb, expressed by the same neurons. Surprisingly, prior studies have shown that both receptors stimulate cAMP accumulation, presenting an enigma of how mushroom body neurons distinguish between acquisition and forgetting signals. Here, we surveyed the spectrum of G protein coupling of dDA1 and Damb and confirmed that both receptors can couple to Gs to stimulate cAMP synthesis. However, the Damb receptor uniquely activates Gq to mobilize Ca2+ signaling with greater efficiency and dopamine sensitivity. The knockdown of Gαq with RNAi in the mushroom bodies inhibits forgetting but has no effect on acquisition. Our findings identify a novel, Damb/Gq signaling pathway that stimulates forgetting, and resolves the opposing effects of dopamine on acquisition and forgetting.

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“…Agonist-dependent cellular measurements of BRET between Venus-Gβ1γ2 and masGRK3ct-Rluc8 or masGRK3ct-Nluc were performed as described previously (80) to examine the activation of G protein signaling in live cells. Sixteen to twenty-four hours after transfection, HEK 293T/17 cells were washed once with phosphate-buffered saline (PBS) containing 5 mM EDTA (EDTA/PBS), and were detached by incubation in EDTA/PBS at room temperature for 5 min.…”

Section: Methodsmentioning

confidence: 99%

Distinct profiles of functional discrimination among G proteins determine the actions of G protein–coupled receptors

et al. 2015

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Members of the G protein coupled receptor (GPCR) family play key roles in many physiological functions and have been extensively exploited pharmacologically to treat diseases. Individual GPCRs exert diverse and distinct effects on cellular physiology and transduce signals by activating heterotrimeric G proteins. Mammalian genomes encode 16 different G protein alpha subunits, and each one of them has distinct properties. Here, we developed a single-platform, optical strategy for the direct monitoring of G protein activation in live cells, and using it we profiled the activities of individual GPCRs across a range of different G proteins, simultaneously quantifying both magnitude of their signaling and activation rates. We report that GPCRs engage multiple G proteins with varying efficacy and kinetics, generating fingerprint-like profiles that define individual receptors. We found that different classes of GPCR ligands, including full and partial agonists, allosteric modulators, and antagonists distinctly affected these fingerprints to functionally bias GPCR signaling. Finally, we showed that intracellular signaling modulators further altered the G protein–coupling profiles of GPCRs, which suggests that their differential expression may alter signaling outcomes in a cell-specific manner. . These observations suggest that the diversity of the effects of GPCRs on cellular physiology may be determined by their differential engagement of multiple G proteins with varying signal magnitudes and activation kinetics, properties that may be exploited pharmacologically.

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Monitoring G Protein Activation in Cells with BRET

Cited by 76 publications

References 9 publications

Screening of GNAL variants in Brazilian patients with isolated dystonia reveals a novel mutation with partial loss of function

Screening of GNAL variants in Brazilian patients with isolated dystonia reveals a novel mutation with partial loss of function

Dopamine Receptor DAMB Signals via Gq to Mediate Forgetting in Drosophila

Distinct profiles of functional discrimination among G proteins determine the actions of G protein–coupled receptors

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