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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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Concentration–response curves for Y1–YFP receptor internalization and Y1 receptor–β-arrestin2 BiFC. Responses to different ligands were measured by granularity analysis applied to automated plate reader images (calculating vesicle average intensity/cell), and normalized to the positive control on each plate (1 µM NPY). Combined data from stably transfected cells are presented for Y1–YFP internalization following 15 min treatment (A, n= 3–5), or Y1 receptor–β-arrestin2 BiFC measured at 60 min, using Yn and Yc partners N173:C155 (B, n= 4–10), N155: C155 (C, n= 4–6) or N173:C173 (D, n= 2–6). Peptides examined included [Leu31, Pro34]NPY (ProNPY) and GR231118 (GR, A and B only). Where appropriate, pEC50 values from the pooled data are presented in Table 1.
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fig05: Concentration–response curves for Y1–YFP receptor internalization and Y1 receptor–β-arrestin2 BiFC. Responses to different ligands were measured by granularity analysis applied to automated plate reader images (calculating vesicle average intensity/cell), and normalized to the positive control on each plate (1 µM NPY). Combined data from stably transfected cells are presented for Y1–YFP internalization following 15 min treatment (A, n= 3–5), or Y1 receptor–β-arrestin2 BiFC measured at 60 min, using Yn and Yc partners N173:C155 (B, n= 4–10), N155: C155 (C, n= 4–6) or N173:C173 (D, n= 2–6). Peptides examined included [Leu31, Pro34]NPY (ProNPY) and GR231118 (GR, A and B only). Where appropriate, pEC50 values from the pooled data are presented in Table 1.

Mentions: NPY concentration–response curves measured in Y1/βarr2 cells yielded similar potency estimates when incubation times were varied from 15 min to 4 h (pEC50 range: 8.39–8.60; n= 3); however, the maximum agonist-induced BiFC response was greatest following incubation times of 1 h or greater. Using 1 h incubation times, comparison of different peptide agonists demonstrated typical Y1 receptor pharmacology for stimulation of Y1/βarr2 BiFC. The endogenous ligands NPY and PYY, and the selective analogue [Leu31, Pro34]NPY were equipotent full agonists (Figure 5). In contrast, the third NPY family member, pancreatic polypeptide (PP), and the metabolite NPY3–36 were much less potent. Both the rank order of potency and the actual pEC50 values obtained were similar to those measured for stimulation of Y1–YFP endocytosis (Figure 5; Table 1). Moreover, concentration–response curves to the different agonists were not influenced by the use of alternative YFP fragment pairs to generate Y1/βarr2 BiFC (Figure 5; Table 1).


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)

Concentration–response curves for Y1–YFP receptor internalization and Y1 receptor–β-arrestin2 BiFC. Responses to different ligands were measured by granularity analysis applied to automated plate reader images (calculating vesicle average intensity/cell), and normalized to the positive control on each plate (1 µM NPY). Combined data from stably transfected cells are presented for Y1–YFP internalization following 15 min treatment (A, n= 3–5), or Y1 receptor–β-arrestin2 BiFC measured at 60 min, using Yn and Yc partners N173:C155 (B, n= 4–10), N155: C155 (C, n= 4–6) or N173:C173 (D, n= 2–6). Peptides examined included [Leu31, Pro34]NPY (ProNPY) and GR231118 (GR, A and B only). Where appropriate, pEC50 values from the pooled data are presented in Table 1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2901518&req=5

fig05: Concentration–response curves for Y1–YFP receptor internalization and Y1 receptor–β-arrestin2 BiFC. Responses to different ligands were measured by granularity analysis applied to automated plate reader images (calculating vesicle average intensity/cell), and normalized to the positive control on each plate (1 µM NPY). Combined data from stably transfected cells are presented for Y1–YFP internalization following 15 min treatment (A, n= 3–5), or Y1 receptor–β-arrestin2 BiFC measured at 60 min, using Yn and Yc partners N173:C155 (B, n= 4–10), N155: C155 (C, n= 4–6) or N173:C173 (D, n= 2–6). Peptides examined included [Leu31, Pro34]NPY (ProNPY) and GR231118 (GR, A and B only). Where appropriate, pEC50 values from the pooled data are presented in Table 1.
Mentions: NPY concentration–response curves measured in Y1/βarr2 cells yielded similar potency estimates when incubation times were varied from 15 min to 4 h (pEC50 range: 8.39–8.60; n= 3); however, the maximum agonist-induced BiFC response was greatest following incubation times of 1 h or greater. Using 1 h incubation times, comparison of different peptide agonists demonstrated typical Y1 receptor pharmacology for stimulation of Y1/βarr2 BiFC. The endogenous ligands NPY and PYY, and the selective analogue [Leu31, Pro34]NPY were equipotent full agonists (Figure 5). In contrast, the third NPY family member, pancreatic polypeptide (PP), and the metabolite NPY3–36 were much less potent. Both the rank order of potency and the actual pEC50 values obtained were similar to those measured for stimulation of Y1–YFP endocytosis (Figure 5; Table 1). Moreover, concentration–response curves to the different agonists were not influenced by the use of alternative YFP fragment pairs to generate Y1/βarr2 BiFC (Figure 5; Table 1).

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