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

Kilpatrick LE, Briddon SJ, Hill SJ, Holliday ND - Br. J. Pharmacol. (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Signalling, School of Biomedical Sciences, University of Nottingham, Queen's Medical Centre, Nottingham, UK.

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.

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Antagonists inhibit Y1 receptor–β-arrestin2 BiFC. In the example, triplicate experiments shown, Y1/βarr2 cells were pretreated for 30 min with antagonists BIBP3226 (A), BIBO3304 (B) and GR231118 (C) at the concentrations indicated. NPY was then applied for 60 min, before measuring BiFC responses (vesicle average intensity/cell, normalized to control 1 µM NPY in each case). NPY concentration–response curves in the absence or presence of antagonist were fitted (GraphPad Prism) assuming shared minimum, maximum and Hill slope constants. The EC50 concentration ratios (CR) were used to construct Schild plots with the following fits for BIBP3326 (pA2 7.8, slope 1.1), BIBO3304 (pA2 9.1, slope 1.0) and GR231118 (pA2 8.3, slope 1.9). Pooled pA2 values are given in the text.
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fig06: Antagonists inhibit Y1 receptor–β-arrestin2 BiFC. In the example, triplicate experiments shown, Y1/βarr2 cells were pretreated for 30 min with antagonists BIBP3226 (A), BIBO3304 (B) and GR231118 (C) at the concentrations indicated. NPY was then applied for 60 min, before measuring BiFC responses (vesicle average intensity/cell, normalized to control 1 µM NPY in each case). NPY concentration–response curves in the absence or presence of antagonist were fitted (GraphPad Prism) assuming shared minimum, maximum and Hill slope constants. The EC50 concentration ratios (CR) were used to construct Schild plots with the following fits for BIBP3326 (pA2 7.8, slope 1.1), BIBO3304 (pA2 9.1, slope 1.0) and GR231118 (pA2 8.3, slope 1.9). Pooled pA2 values are given in the text.

Mentions: A peptide Y1 receptor antagonist GR231118, reported to increase Y1 receptor internalization (Pheng et al., 2003), was inactive in both Y1–YFP endocytosis and βarr2 BiFC assays (Figure 5). Equally, Y1/βarr2 BiFC was not altered by the non-peptide antagonists BIBP3226 (1 µM yielded −4.5 ± 10.0% of 1 µM NPY response; n= 4) or BIBO3304 (at 30 nM: −8.7 ± 7.5% of 1 µM NPY response; n= 5). However, 30 min pre-incubation with either BIBP3226 or BIBO3304 resulted in parallel rightward shifts of the NPY concentration–response curves for Y1/βarr2 BiFC, consistent with competitive reversible antagonism (Figure 6). The respective Schild plots yielded pA2 estimates of 8.0 ± 0.1 for BIBP3226 (slope 1.1 ± 0.1, n= 4) and 9.0 ± 0.1 for BIBO3304 (slope 1.1 ± 0.1, n= 5). This functional estimate for BIBO3304 affinity was the same as its pKi measured in [125I]PYY competition assays in Y1/βarr2 membranes (Table 2). GR231118 also acted as an antagonist in similar pre-incubation experiments (Figure 6C), yielding a pA2 estimate of 8.6 ± 0.2 (n= 4); however, linear GR231118 Schild plots were greater than unity (slope 1.5 ± 0.2).


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

Kilpatrick LE, Briddon SJ, Hill SJ, Holliday ND - Br. J. Pharmacol. (2010)

Antagonists inhibit Y1 receptor–β-arrestin2 BiFC. In the example, triplicate experiments shown, Y1/βarr2 cells were pretreated for 30 min with antagonists BIBP3226 (A), BIBO3304 (B) and GR231118 (C) at the concentrations indicated. NPY was then applied for 60 min, before measuring BiFC responses (vesicle average intensity/cell, normalized to control 1 µM NPY in each case). NPY concentration–response curves in the absence or presence of antagonist were fitted (GraphPad Prism) assuming shared minimum, maximum and Hill slope constants. The EC50 concentration ratios (CR) were used to construct Schild plots with the following fits for BIBP3326 (pA2 7.8, slope 1.1), BIBO3304 (pA2 9.1, slope 1.0) and GR231118 (pA2 8.3, slope 1.9). Pooled pA2 values are given in the text.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2901518&req=5

fig06: Antagonists inhibit Y1 receptor–β-arrestin2 BiFC. In the example, triplicate experiments shown, Y1/βarr2 cells were pretreated for 30 min with antagonists BIBP3226 (A), BIBO3304 (B) and GR231118 (C) at the concentrations indicated. NPY was then applied for 60 min, before measuring BiFC responses (vesicle average intensity/cell, normalized to control 1 µM NPY in each case). NPY concentration–response curves in the absence or presence of antagonist were fitted (GraphPad Prism) assuming shared minimum, maximum and Hill slope constants. The EC50 concentration ratios (CR) were used to construct Schild plots with the following fits for BIBP3326 (pA2 7.8, slope 1.1), BIBO3304 (pA2 9.1, slope 1.0) and GR231118 (pA2 8.3, slope 1.9). Pooled pA2 values are given in the text.
Mentions: A peptide Y1 receptor antagonist GR231118, reported to increase Y1 receptor internalization (Pheng et al., 2003), was inactive in both Y1–YFP endocytosis and βarr2 BiFC assays (Figure 5). Equally, Y1/βarr2 BiFC was not altered by the non-peptide antagonists BIBP3226 (1 µM yielded −4.5 ± 10.0% of 1 µM NPY response; n= 4) or BIBO3304 (at 30 nM: −8.7 ± 7.5% of 1 µM NPY response; n= 5). However, 30 min pre-incubation with either BIBP3226 or BIBO3304 resulted in parallel rightward shifts of the NPY concentration–response curves for Y1/βarr2 BiFC, consistent with competitive reversible antagonism (Figure 6). The respective Schild plots yielded pA2 estimates of 8.0 ± 0.1 for BIBP3226 (slope 1.1 ± 0.1, n= 4) and 9.0 ± 0.1 for BIBO3304 (slope 1.1 ± 0.1, n= 5). This functional estimate for BIBO3304 affinity was the same as its pKi measured in [125I]PYY competition assays in Y1/βarr2 membranes (Table 2). GR231118 also acted as an antagonist in similar pre-incubation experiments (Figure 6C), yielding a pA2 estimate of 8.6 ± 0.2 (n= 4); however, linear GR231118 Schild plots were greater than unity (slope 1.5 ± 0.2).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Signalling, School of Biomedical Sciences, University of Nottingham, Queen's Medical Centre, Nottingham, UK.

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.

Show MeSH
Related in: MedlinePlus