Measurement of heterotrimeric G-protein and regulators of G-protein signaling interactions by time-resolved fluorescence resonance energy transfer

Leifert, Wayne R.; Bailey, Kelly; Cooper, Thomas J.; Aloia, Amanda L.; Glatz, Richard; McMurchie, Edward J.

doi:10.1016/j.ab.2006.04.042

Cited by 23 publications

(18 citation statements)

References 44 publications

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“…3B). This indicated that a high affinity interaction was occurring between CrV2-Alexa and G␣ i1 -Tb, which was comparable with the affinity between the G␣ subunit and the G␤␥ dimer (2 nM) measured using the same technique (34). To show the TR-FRET signal was attributable to a specific protein/protein interaction, proteinase K, a broad spectrum serine protease that cleaves peptide bonds at the carboxylic sides of aliphatic, aromatic, or hydrophobic amino acids, was used to digest the proteins.…”

Section: Identification Of Crv2supporting

confidence: 59%

“…Specific in Vitro Interaction between CrV2 and G␣-In this study, we used an established in vitro TR-FRET assay (34,35) to identify a novel interaction between the G-protein subunit G␣, and CrV2. We demonstrated that CrV2 binds to G␣ subunits from rat and Drosophila, with nanomolar affinity (K d ϭ 6.2 nM).…”

Section: Discussionmentioning

confidence: 99%

“…Nucleotide exchange causes conformation changes such that the G␣ subunit and the G␤␥ dimer interact with downstream effectors, including various enzymes and ion channels that alter cellular metabolism (33). We previously developed a TR-FRET (time-resolved Förster resonance energy transfer) assay utilizing a terbium-chelate donor and Alexa546 acceptor to demonstrate interactions of the G␣ subunit with the G␤␥ dimer and the regulator of G-protein signaling 4 (RGS4) (34). The assay was recently adapted by others to monitor the effect of allosteric modulators of RGS4 in altering RGS4:G␣ affinity and further developed into a high throughput screen for other such modulators (35).…”

mentioning

confidence: 99%

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Identification of an in Vitro Interaction between an Insect Immune Suppressor Protein (CrV2) and Gα Proteins

Cooper

Bailey-Hill

Leifert

et al. 2011

Journal of Biological Chemistry

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The protein CrV2 is encoded by a polydnavirus integrated into the genome of the endoparasitoid Cotesia rubecula (Hymenoptera:Braconidae:Microgastrinae) and is expressed in host larvae with other gene products of the polydnavirus to allow successful development of the parasitoid. CrV2 expression has previously been associated with immune suppression, although the molecular basis for this was not known. Here, we have used time-resolved Förster resonance energy transfer (TR-FRET) to demonstrate high affinity binding of CrV2 to G␣ subunits (but not the G␤␥ dimer) of heterotrimeric G-proteins. Signals up to 5-fold above background were generated, and an apparent dissociation constant of 6.2 nM was calculated. Protease treatment abolished the TR-FRET signal, and the presence of unlabeled CrV2 or G␣ proteins also reduced the TR-FRET signal. The activation state of the G␣ subunit was altered with aluminum fluoride, and this decreased the affinity of the interaction with CrV2. It was also demonstrated that CrV2 preferentially bound to Drosophila G␣ o compared with rat G␣ i1 . In addition, three CrV2 homologs were detected in sequences derived from polydnaviruses from Cotesia plutellae and Cotesia congregata (including the immune-related early expressed transcript, EP2). These data suggest a potential mode-of-action of immune suppressors not previously reported, which in addition to furthering our understanding of insect immunity may have practical benefits such as facilitating development of novel controls for pest insect species.

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Section: Identification Of Crv2supporting

confidence: 59%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Identification of an in Vitro Interaction between an Insect Immune Suppressor Protein (CrV2) and Gα Proteins

Cooper

Bailey-Hill

Leifert

et al. 2011

Journal of Biological Chemistry

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“…TR-FRET experiments were performed on a PHERAstar multipurpose microplate reader (BMG Labtech GmbH, Offenberg, Germany) using the LanthaScreen filter set. These experiments were based on the method of Leifert et al (2006). For the saturation experiments, Tb-G␣ o was diluted to 20 nM in 50 mM HEPES, pH 8.0, 100 mM NaCl, 0.1% Lubrol, 30 M GDP, 5 mM NaF, 5 mM MgCl2, and 5 M AlCl 3 and allowed to activate for 10 min on ice before use.…”

Section: Methodsmentioning

confidence: 99%

Reversible, Allosteric Small-Molecule Inhibitors of Regulator of G Protein Signaling Proteins

Blazer¹,

Roman²,

Chung³

et al. 2010

Mol Pharmacol

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Regulators of G protein signaling (RGS) proteins are potent negative modulators of G protein signaling and have been proposed as potential targets for small-molecule inhibitor development. We report a high-throughput time-resolved fluorescence resonance energy transfer screen to identify inhibitors of RGS4 and describe the first reversible small-molecule inhibitors of an RGS protein. Two closely related compounds, typified by CCG-63802 [((, inhibit the interaction between RGS4 and G␣ o with an IC 50 value in the low micromolar range. They show selectivity among RGS proteins with a potency order of RGS 4 Ͼ 19 ϭ 16 Ͼ 8 Ͼ Ͼ 7. The compounds inhibit the GTPase accelerating protein activity of RGS4, and thermal stability studies demonstrate binding to the RGS but not to G␣ o . On RGS4, they depend on an interaction with one or more cysteines in a pocket that has previously been identified as an allosteric site for RGS regulation by acidic phospholipids. Unlike previous small-molecule RGS inhibitors identified to date, these compounds retain substantial activity under reducing conditions and are fully reversible on the 10-min time scale. CCG-63802 and related analogs represent a useful step toward the development of chemical tools for the study of RGS physiology.

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“…The first published small molecule RGS inhibitor, CCG-4986, was identified by the group of Richard Neubig, using a novel flow cytometry-based PPI assay (Roman et al, 2007). Subsequent work from the same group used biochemical time-resolved FRET (TR-FRET) (Leifert et al, 2006) to identify CCG-63802, and analogues thereof, as the first reversible RGS protein inhibitor . Like CCG-4986, CCG-63802 showed selectivity for RGS4 over other RGS proteins studied.…”

Section: Advances In Rgs Protein Drug Discovery -From Biochemical Actmentioning

confidence: 99%

The evolution of regulators of G protein signalling proteins as drug targets – 20 years in the making: IUPHAR Review 21

Sjögren

2017

British J Pharmacology

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Regulators of G protein signalling (RGS) proteins are celebrating the 20th anniversary of their discovery. The unveiling of this new family of negative regulators of G protein signalling in the mid-1990s solved a persistent conundrum in the G protein signalling field, in which the rate of deactivation of signalling cascades in vivo could not be replicated in exogenous systems. Since then, there has been tremendous advancement in the knowledge of RGS protein structure, function, regulation and their role as novel drug targets. RGS proteins play an important modulatory role through their GTPase-activating protein (GAP) activity at active, GTP-bound Gα subunits of heterotrimeric G proteins. They also possess many non-canonical functions not related to G protein signalling. Here, an update on the status of RGS proteins as drug targets is provided, highlighting advances that have led to the inclusion of RGS proteins in the IUPHAR/BPS Guide to PHARMACOLOGY database of drug targets.Abbreviations DEP, Disheveled Egl-10 Pleckstrin; GAP, GTPase-activating protein; GGL, G protein γ-like; PPI, protein-protein interaction; RGS, regulator of G protein signalling; R7BP, R7 binding protein; R9AP, RGS9 associated protein IntroductionG protein-mediated signalling pathways have played a pivotal role in drug discovery and development for many decades. The large family of GPCRs or their downstream effectors are the target of 40% of clinically used drugs and thus represent a multi-billion-dollar industry (Wise et al., 2002). Interestingly, of the more than 300 non-olfactory GPCRs known, only a fraction of them are targeted by drugs. Thus, there is a large untapped area of drug development still available. Moreover, many GPCR drugs are associated with low efficacy and/or side effects. More targeted therapies are therefore required, and as we learn more about the structure and function of GPCRs and their regulators, these goals will be achievable.All biological signals are tightly regulated and for every on-switch there is usually an off-switch. GPCRs are activated by ligands, transmitting signalling information to Gα subunits of heterotrimeric G proteins by enhancing the exchange of GDP for GTP in the Gα nucleotide binding site, which results in the dissociation of Gα from Gβγ dimers and activation of both G protein components. Deactivation of G proteins does not occur by simple reversal of nucleotide exchange, but rather by an independently regulated GTPase activity, hydrolyzing GTP to GDP. Although Gα proteins possess an intrinsic ability to hydrolyze GTP, this process is very slow and cannot account for the transient nature of intracellular signalling cascades in vivo. Hence, additional kinetic mechanisms are required for the physiological timing of signals. One of the most critical of these kinetic mechanisms is mediated through regulator of G protein signalling (RGS) proteins, which have received increasing interest as novel drug targets in the past two decades. As a result, RGS proteins have now been added as the most recent ad...

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Measurement of heterotrimeric G-protein and regulators of G-protein signaling interactions by time-resolved fluorescence resonance energy transfer

Cited by 23 publications

References 44 publications

Identification of an in Vitro Interaction between an Insect Immune Suppressor Protein (CrV2) and Gα Proteins

Identification of an in Vitro Interaction between an Insect Immune Suppressor Protein (CrV2) and Gα Proteins

Reversible, Allosteric Small-Molecule Inhibitors of Regulator of G Protein Signaling Proteins

The evolution of regulators of G protein signalling proteins as drug targets – 20 years in the making: IUPHAR Review 21

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