Quantitative analysis of neuropeptide Y receptor association with β‐arrestin2 measured by bimolecular fluorescence complementation

摘要: 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) β-arrestin2, using bimolecular fluorescence complementation (BiFC) to directly report underlying protein–protein interactions. Experimental approach:  Y1 were tagged with a C-terminal fragment, Yc, of yellow fluorescent protein (YFP), β-arrestin2 fused complementary N-terminal Yn. After receptor–β-arrestin association, YFP fragment refolding regenerate was examined by confocal microscopy in transfected HEK293 cells. receptor/β-arrestin2 BiFC responses also quantified automated imaging granularity analysis. Key results:  NPY stimulation promoted Y1–Yc β-arrestin2–Yn, specific development intracellular compartments, eliminated when non-interacting receptor arrestin mutants. Responses developed irreversibly slower than for downstream receptor–YFP internalization, consequence delayed maturation stability complemented YFP. However, measurements delivered appropriate ligand pharmacology both Y2 receptors, demonstrated higher affinity compared β-arrestin2. Receptor mutagenesis combined revealed alternative arrangements Ser/Thr residues C tail could support receptor–β-arrestin2 interaction enhanced loop mutation H155P. Conclusions implications:  The approach quantifies focused on pathway, provides insight into mechanisms recruitment activated phosphorylated 7TMRs, at level interaction.