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• W1494263850 startingPage "892" @default.
• W1494263850 abstract "beta-Arrestins are critical scaffold proteins that shape spatiotemporal signalling from seven transmembrane domain receptors (7TMRs). Here, we study the association between neuropeptide Y (NPY) receptors and beta-arrestin2, using bimolecular fluorescence complementation (BiFC) to directly report underlying protein-protein interactions.Y1 receptors were tagged with a C-terminal fragment, Yc, of yellow fluorescent protein (YFP), and beta-arrestin2 fused with the complementary N-terminal fragment, Yn. After Y receptor-beta-arrestin association, YFP fragment refolding to regenerate fluorescence (BiFC) was examined by confocal microscopy in transfected HEK293 cells. Y receptor/beta-arrestin2 BiFC responses were also quantified by automated imaging and granularity analysis.NPY stimulation promoted association between Y1-Yc and beta-arrestin2-Yn, and the specific development of BiFC in intracellular compartments, eliminated when using non-interacting receptor and arrestin mutants. Responses developed irreversibly and were slower than for downstream Y1 receptor-YFP internalization, a consequence of delayed maturation and stability of complemented YFP. However, beta-arrestin2 BiFC measurements delivered appropriate ligand pharmacology for both Y1 and Y2 receptors, and demonstrated higher affinity of Y1 compared to Y2 receptors for beta-arrestin2. Receptor mutagenesis combined with beta-arrestin2 BiFC revealed that alternative arrangements of Ser/Thr residues in the Y1 receptor C tail could support beta-arrestin2 association, and that Y2 receptor-beta-arrestin2 interaction was enhanced by the intracellular loop mutation H155P.The BiFC approach quantifies Y receptor ligand pharmacology focused on the beta-arrestin2 pathway, and provides insight into mechanisms of beta-arrestin2 recruitment by activated and phosphorylated 7TMRs, at the level of protein-protein interaction." @default.
• W1494263850 created "2016-06-24" @default.
• W1494263850 creator A5009862868 @default.
• W1494263850 creator A5051941945 @default.
• W1494263850 creator A5057506128 @default.
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• W1494263850 date "2010-04-28" @default.
• W1494263850 modified "2023-09-30" @default.
• W1494263850 title "Quantitative analysis of neuropeptide Y receptor association with β-arrestin2 measured by bimolecular fluorescence complementation" @default.
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• W1494263850 doi "https://doi.org/10.1111/j.1476-5381.2010.00676.x" @default.
• W1494263850 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2901518" @default.
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