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


METHODS
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.


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
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.
Kilpatrick
L E
LE
Institute of Cell Signalling, School of Biomedical Sciences, University of Nottingham, Queen's Medical Centre, Nottingham, UK.
Briddon
S J
SJ
Hill
S J
SJ
Holliday
N D
ND
D019390:Arrestins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods; D050505:Mutant Proteins / Q000378:metabolism; D009478:Neuropeptide Y / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism; D019894:Peptide YY / Q000378:metabolism; D010766:Phosphorylation / Q000235:genetics; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods*; D017476:Receptors, Neuropeptide Y / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000071557:beta-Arrestins
D019390:Arrestins; D008024:Ligands; D008164:Luminescent Proteins; D050505:Mutant Proteins; D009478:Neuropeptide Y; D017476:Receptors, Neuropeptide Y; D011993:Recombinant Fusion Proteins; D000071557:beta-Arrestins; C097212:neuropeptide Y-Y1 receptor; C095889:neuropeptide Y2 receptor; D019894:Peptide YY