Dataset Information

A Split Luciferase Complementation Assay for the Quantification of ?-Arrestin2 Recruitment to Dopamine D2-Like Receptors.

ABSTRACT: Investigations on functional selectivity of GPCR ligands have become increasingly important to identify compounds with a potentially more beneficial side effect profile. In order to discriminate between individual signaling pathways, the determination of ?-arrestin2 recruitment, in addition to G-protein activation, is of great value. In this study, we established a sensitive split luciferase-based assay with the ability to quantify ?-arrestin2 recruitment to D2long and D3 receptors and measure time-resolved ?-arrestin2 recruitment to the D2long receptor after agonist stimulation. We were able to characterize several standard (inverse) agonists as well as antagonists at the D2longR and D3R subtypes, whereas for the D4.4R, no ?-arrestin2 recruitment was detected, confirming previous reports. Extensive radioligand binding studies and comparisons with the respective wild-type receptors confirm that the attachment of the Emerald luciferase fragment to the receptors does not affect the integrity of the receptor proteins. Studies on the involvement of GRK2/3 and PKC on the ?-arrestin recruitment to the D2longR and D3R, as well as at the D1R using different kinase inhibitors, showed that the assay could also contribute to the elucidation of signaling mechanisms. Its broad applicability, which provides concentration-dependent and kinetic information on receptor/?-arrestin2 interactions, renders this homogeneous assay a valuable method for the identification of biased agonists.

SUBMITTER: Forster L

PROVIDER: S-EPMC7503597 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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A Split Luciferase Complementation Assay for the Quantification of β-Arrestin2 Recruitment to Dopamine D2-Like Receptors.

Forster Lisa L Grätz Lukas L Mönnich Denise D Bernhardt Günther G Pockes Steffen S

International journal of molecular sciences 20200824 17

Investigations on functional selectivity of GPCR ligands have become increasingly important to identify compounds with a potentially more beneficial side effect profile. In order to discriminate between individual signaling pathways, the determination of β-arrestin2 recruitment, in addition to G-protein activation, is of great value. In this study, we established a sensitive split luciferase-based assay with the ability to quantify β-arrestin2 recruitment to D2long and D3 r ...[more]

PMID: 32847148

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Project description:BackgroundIP3-induced Ca2+ release, mediated by IP3R, is one of the most momentous cellular signaling mechanisms that regulate in a wide variety of essential cellular functions. Involvement of disrupted IP3 signaling pathways in numerous pathophysiology conditions is implicated to find the best methods for its measurement. Hence, several different biosensors have developed to monitor temporal changes of IP3 by using the IP3-binding domain of IP3 receptors.ObjectivesBased on a previous study, we developed and characterized a series of bioluminescent biosensors using the human type-II IP3 receptor ligand binding domain (residues 1-604), named LAIRE (luminescent analyzer for IP3 receptor element) to study the effect of flexible and rigid linkers on the luminescence intensity of split luciferase. The effect of a mutation in IP3 binding residues and suppressor domain in the IP3 binding domain on luciferase complementary assay is also investigated.Materials and methodsIn the present study, first IP3-binding domain (residues 1-604) of IP3-receptor type 2 (LAIRE) was fused between complementary non-functional fragments of firefly luciferase and then the rigid linker sequence (LLRAIEAQQHLL), selected by ProDA database, introduced between Nluc and ligand binding domain and compared with that of the flexible linker ((GGGGS)2) in LAIRE chimera. The IP3-insensitive mutant of the biosensor was constructed using the Stratagene QuikChange® procedure. In order to the analysis of the dynamical movements of selected structures in the large-scale, coarse-graining method of molecular dynamics simulation (1µs) was applied.ResultsAs expected, the flexible linker brings two inactive fragments of luciferase together relative to the rigid linker and leads to complementation of luciferase activity, which is detected using luciferin. However, this conformational flexibility in linker increases background to noise ratio and attenuates fold induction.ConclusionsIt seems that the ligand binding properties of IP3 binding core make it more suitable for the design of biosensor than the ligand binding domain.

Dataset Information

A Split Luciferase Complementation Assay for the Quantification of ?-Arrestin2 Recruitment to Dopamine D2-Like Receptors.

Publications

A Split Luciferase Complementation Assay for the Quantification of β-Arrestin2 Recruitment to Dopamine D<sub>2</sub>-Like Receptors.

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