Quantitative analysis of neuropeptide Y receptor association with beta-arrestin2 measured by bimolecular fluorescence complementation

Br J Pharmacol. 2010 Jun;160(4):892-906. doi: 10.1111/j.1476-5381.2010.00676.x. Epub 2010 Apr 28.

Abstract

Background and purpose: 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.

Experimental approach: 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.

Key results: 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.

Conclusions and implications: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arrestins / genetics
  • Arrestins / metabolism*
  • Cell Line
  • Humans
  • Image Processing, Computer-Assisted
  • Kinetics
  • Ligands
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Microscopy, Confocal
  • Microscopy, Fluorescence / methods
  • Mutant Proteins / metabolism
  • Neuropeptide Y / agonists
  • Neuropeptide Y / antagonists & inhibitors
  • Neuropeptide Y / metabolism
  • Peptide YY / metabolism
  • Phosphorylation / genetics
  • Protein Interaction Domains and Motifs
  • Protein Interaction Mapping / methods*
  • Receptors, Neuropeptide Y / agonists
  • Receptors, Neuropeptide Y / antagonists & inhibitors
  • Receptors, Neuropeptide Y / genetics
  • Receptors, Neuropeptide Y / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • beta-Arrestins

Substances

  • Arrestins
  • Ligands
  • Luminescent Proteins
  • Mutant Proteins
  • Neuropeptide Y
  • Receptors, Neuropeptide Y
  • Recombinant Fusion Proteins
  • beta-Arrestins
  • neuropeptide Y-Y1 receptor
  • neuropeptide Y2 receptor
  • Peptide YY