Dataset Information

?-arrestin2 recruitment at the ?2 adrenergic receptor: A luciferase complementation assay adapted for undergraduate training in pharmacology.

ABSTRACT: In the context of pharmacology teaching, hands-on activities constitute an essential complement to theoretical lectures. Frequently, these activities consist in exposing fresh animal tissues or even living animals to selected drugs and qualitatively or quantitatively evaluating functional responses. However, technological advancements in pharmacological research and the growing concerns for animal experimentation support the need for innovative and flexible in vitro assays adapted for teaching purposes. We herein report the implementation of a luciferase complementation assay (LCA) enabling to dynamically monitor ?-arrestin2 recruitment at the ?₂ adrenergic receptor in the framework of pharmacological training at the faculty of Pharmacy and Biomedical Sciences. The assay allowed students to quantitatively characterize the competitive antagonism of propranolol, and to calculate pEC₅₀ , pK_B , and pA₂ values after a guided data analysis session. Moreover, the newly implemented workshop delivered highly reproducible results and were generally appreciated by students. As such, we report that the luciferase complementation-based assay proved to be a straightforward, robust, and cost-effective alternative to experiments performed on animal tissues, constituting a useful and flexible tool to enhance and update current hands-on training in the context of pharmacological teaching.

SUBMITTER: Ferraiolo M

PROVIDER: S-EPMC7842874 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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β-arrestin2 recruitment at the β2 adrenergic receptor: A luciferase complementation assay adapted for undergraduate training in pharmacology.

Ferraiolo Mattia M Beckers Pauline P Marquet Nicolas N Roumain Martin M Ruiz Lucie L Dupuis Nadine N Hanson Julien J Hermans Emmanuel E

Pharmacology research & perspectives 20210201 1

In the context of pharmacology teaching, hands-on activities constitute an essential complement to theoretical lectures. Frequently, these activities consist in exposing fresh animal tissues or even living animals to selected drugs and qualitatively or quantitatively evaluating functional responses. However, technological advancements in pharmacological research and the growing concerns for animal experimentation support the need for innovative and flexible in vitro assays adapted for teaching p ...[more]

PMID: 33508174

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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.

Project description:The regulation of organismic lipid storage amounts is a central goal of all organisms. Proteins associated with the dedicated lipid storage organelles, the so-called lipid droplets (LDs), are instrumental to this regulation. LD proteomes of diverse organisms have been characterized by mass spectrometry. Yet, the functional characterization of most of these proteins is still lagging behind. Moreover, knowledge of interactions among LD-associated proteins is sparse and mostly limited to proteins involved in mammalian lipolysis regulation. This process crucially depends on an orchestrated set of protein-protein interactions which limit the accessibility, and thus remobilization, of the lipid stores. Thus, a better knowledge of the LD-associated protein interactome likely reveals entry points to deeper understand lipid storage regulation on a mechanistic level. Here, we utilize a split-luciferase based protein-protein interaction assay to identify interactions among 46 LD-associated proteins and protein variants of which 40 are Drosophila and 6 mouse proteins. In 1531 complementation tests, we assayed for interactions among 487 luciferase fragment fusion protein pairs (covered by 830 different construct combinations) of which 85 (17.45 %) resulted in a significant luciferase complementation. These results suggest, that a prominent fraction of the LD-associated proteome participates in protein-protein interactions, and provide a basis for further functional studies. Besides confirming previously described interactions, we identified several new protein interaction pairs. Of these, we characterize the following three in greater detail: (i) Jabba and CG9186, (ii) Jabba and Histones and (iii) Ubiquitin and CG9186. The interaction among Jabba and Histones is of physiological significance and we provide data suggesting that Jabba alone is sufficient to recruit Histones to LDs. Further, we identified a positively charged amino acid stretch of 16 amino acids within the Jabba protein which is necessary for the Jabba-Histone interaction. CG9186 is an annotated esterase which is involved in lipid storage amount regulation and positioning of LDs. We present data supporting the ubiquitination of at least two lysine residues of CG9186 and demonstrate a role of CG9186 ubiquitination in the induction of LD clusters linked to the overexpression of CG9186.

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

?-arrestin2 recruitment at the ?2 adrenergic receptor: A luciferase complementation assay adapted for undergraduate training in pharmacology.

Publications

β-arrestin2 recruitment at the β2 adrenergic receptor: A luciferase complementation assay adapted for undergraduate training in pharmacology.

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