Quantitative analysis of neuropeptide Y receptor association with beta-arrestin2 measured by bimolecular fluorescence complementation
| Autor: | L E, Kilpatrick, S J, Briddon, S J, Hill, N D, Holliday |
|---|---|
| Rok vydání: | 2010 |
| Předmět: |
Microscopy
Confocal neuropeptide Y arrestin Arrestins Recombinant Fusion Proteins desensitization Ligands Research Papers Receptors Neuropeptide Y Cell Line internalization Kinetics Luminescent Proteins Microscopy Fluorescence seven transmembrane domain receptor Protein Interaction Mapping automated confocal imaging Image Processing Computer-Assisted Humans Mutant Proteins Peptide YY Protein Interaction Domains and Motifs Phosphorylation bimolecular fluorescence complementation beta-Arrestins |
| Zdroj: | British Journal of Pharmacology |
| ISSN: | 1476-5381 |
| Popis: | Background and purpose: β-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 β-arrestin2, using bimolecular fluorescence complementation (BiFC) to directly report underlying protein–protein interactions. Experimental approach: Y1 receptors were tagged with a C-terminal fragment, Yc, of yellow fluorescent protein (YFP), and β-arrestin2 fused with the complementary N-terminal fragment, Yn. After Y receptor–β-arrestin association, YFP fragment refolding to regenerate fluorescence (BiFC) was examined by confocal microscopy in transfected HEK293 cells. Y receptor/β-arrestin2 BiFC responses were also quantified by automated imaging and granularity analysis. Key results: NPY stimulation promoted association between Y1–Yc and β-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, β-arrestin2 BiFC measurements delivered appropriate ligand pharmacology for both Y1 and Y2 receptors, and demonstrated higher affinity of Y1 compared to Y2 receptors for β-arrestin2. Receptor mutagenesis combined with β-arrestin2 BiFC revealed that alternative arrangements of Ser/Thr residues in the Y1 receptor C tail could support β-arrestin2 association, and that Y2 receptor–β-arrestin2 interaction was enhanced by the intracellular loop mutation H155P. Conclusions and implications: The BiFC approach quantifies Y receptor ligand pharmacology focused on the β-arrestin2 pathway, and provides insight into mechanisms of β-arrestin2 recruitment by activated and phosphorylated 7TMRs, at the level of protein–protein interaction. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|