Quantitative analysis of Neuropeptide Y Y1 receptor interaction with β-arrestin2 using bimolecular fluorescence complementation (BiFC)

Nick Holliday, Laura Kilpatrick, Stephen Briddon, Stephen Hill

University of Nottingham, Nottingham, UK

When N and C terminal fragments of venus yellow fluorescent protein (YFP) are brought together by specific interaction of tagged proteins, complementation restores YFP structure and fluorescence (BiFC). So far this approach has been most valuable in probing stable protein-protein complexes (Kerppola, 2008). Here we demonstrate that BiFC can also quantify agonist-stimulated association between a G protein coupled receptor (the Neuropeptide Y (NPY) Y1 subtype) and βarrestin2.

Stable HEK293T cells expressed Y1 receptor-YFP, or both Y1-YC(155-238 fragment) and βarrestin2-YN(1-173), called Y1 / βarr2. Confluent cells on Costar 3904 plates were incubated for 30 min at 37°C in DMEM / 0.1% BSA, ± Y1 antagonist BIBP3226 (Rudolf et al., 1994), before agonist was added to the same media. After 3 % PFA fixation and nuclear staining with H33342, images were automatically acquired on an IX Ultra platereader (Molecular Devices). Granularity analysis of Y1-YFP internalisation and Y1 / βarr2 BiFC quantified average intensity of 3 – 18 μm perinuclear vesicles, normalised to plate controls (vehicle / 1 μM NPY). Pooled concentration-response data was analysed by Graphpad Prism v5.

100 nM NPY rapidly increased BiFC YFP fluorescence in Y1 / βarr2 cells (t1/2 10.4 ± 1.0 min, n = 4). The NPY pEC50 value at 60 min was similar to the Y1 agonists peptide YY and [Leu31, Pro34]NPY, while NPY3-36 and pancreatic polypeptide (PP) were less potent (Table 1). BIBP3226 inhibited NPY stimulated Y1/ βarr2 BiFC with a pA2 of 8.0 ± 0.1 (Schild slope 1.1 ± 0.1, n = 4). Similar agonist pEC50 values were obtained for Y1-YFP internalisation at 15 min (Table 1), but 100nM NPY induced endocytosis was faster than the BiFC response (t1/2 2.4 ± 0.3 min, n = 3, P < 0.01 Student&apos;s t test), and was reversed after 60 min wash (8.3 ± 11.7 % of non-washed NPY response; n = 3). In contrast NPY induced BiFC in Y1 / βarr2 cells was irreversible under the same conditions (100 nM; 75.4 ± 12.0 % of non-washed NPY, n = 4; P < 0.05 compared to Y1-YFP). Thus quantitative BiFC measures Y1 receptor association with βarrestin2, and allows assessment of ligand pharmacology through this signalling pathway. This approach should also prove useful in mutational analysis of receptor-arrestin interactions (Kilpatrick et al., this meeting).

We thank the MRC for financial support and Dr. F. Ciruela for the original YFP BiFC constructs.

Kerppola, TK (2008) Ann.Rev.Biophys. 37, 465. Rudolf, K et al. (1994) Eur.J.Pharmacol. 271, R11.