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Luciferase complementation based-detection of G-protein-coupled receptor activity.

ABSTRACT: Protein complementation assays (PCA) are used as pharmacological tools, enabling a wide array of applications, ranging from studies of protein-protein interactions to second messenger effects. Methods to detect activities of G protein-coupled receptors (GPCRs) have particular relevance for drug screening. Recent development of an engineered luciferase NanoLuc created the possibility of generating a novel PCA, which in turn could open a new avenue for developing drug screening assays. Here we identified a novel split position for NanoLuc and demonstrated its use in a series of fusion constructs to detect the activity of GPCRs. The split construct can be applied to a variety of pharmacological screening systems.

SUBMITTER: Yano H

PROVIDER: S-EPMC7365683 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Luciferase complementation based-detection of G-protein-coupled receptor activity.

Yano Hideaki H Cai Ning Sheng NS Javitch Jonathan A JA Ferré Sergi S

BioTechniques 20180701 1

Protein complementation assays (PCA) are used as pharmacological tools, enabling a wide array of applications, ranging from studies of protein-protein interactions to second messenger effects. Methods to detect activities of G protein-coupled receptors (GPCRs) have particular relevance for drug screening. Recent development of an engineered luciferase NanoLuc created the possibility of generating a novel PCA, which in turn could open a new avenue for developing drug screening assays. Here we ide ...[more]

PMID: 30014734

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Project description:A critical challenge for all organisms is to carefully control the amount of lipids they store. An important node for this regulation is the protein coat present at the surface of lipid droplets (LDs), the intracellular organelles dedicated to lipid storage. Only limited aspects of this regulation are understood so far. For the probably best characterized case, the regulation of lipolysis in mammals, some of the major protein players have been identified, and it has been established that this process crucially depends on an orchestrated set of protein-protein interactions. Proteomic analysis has revealed that LDs are associated with dozens, if not hundreds, of different proteins, most of them poorly characterized, with even fewer data regarding which of them might physically interact. To comprehensively understand the mechanism of lipid storage regulation, it will likely be essential to define the interactome of LD-associated proteins.Previous studies of such interactions were hampered by technical limitations. Therefore, we have developed a split-luciferase based protein-protein interaction assay and test for interactions among 47 proteins from Drosophila and from mouse. We confirmed previously described interactions and identified many new ones. In 1561 complementation tests, we assayed for interactions among 487 protein pairs of which 92 (19%) resulted in a successful luciferase complementation. These results suggest that a prominent fraction of the LD-associated proteome participates in protein-protein interactions.In targeted experiments, we analyzed the two proteins Jabba and CG9186 in greater detail. Jabba mediates the sequestration of histones to LDs. We successfully applied our split luciferase complementation assay to learn more about this function as we were e.g. able to map the interaction between Jabba and histones. For CG9186, expression levels affect the positioning of LDs. Here, we reveal the ubiquitination of CG9186, and link this posttranslational modification to LD cluster induction.

Dataset Information

Luciferase complementation based-detection of G-protein-coupled receptor activity.

Publications

Luciferase complementation based-detection of G-protein-coupled receptor activity.

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