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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.

Show MeSH
Agonist and antagonist data for human Y1 receptor stimulated β-arrestin2 BiFC. Human hY1/βarr2 cells were derived after further dilution cloning to optimize receptor and arrestin construct expression. The similarity with rat Y1/βarr2 cell data is illustrated by agonist concentration–response curves in (A), for 60 min treatment with NPY (pEC50: 8.99 ± 0.08, n= 7), PYY (pEC50: 8.76 ± 0.09, n= 6), PP (n= 6), [Leu31, Pro34]NPY (ProNPY pEC50: 8.65 ± 0.06, n= 7) and NPY3–36 (n= 6). In (B), 30 min pre-incubation with 10 nM BIBO3304 (BIBO) resulted in a 9.1-fold rightward shift in the NPY concentration–response relationship (n= 5) from which a pKB of 8.9 was calculated.
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fig07: Agonist and antagonist data for human Y1 receptor stimulated β-arrestin2 BiFC. Human hY1/βarr2 cells were derived after further dilution cloning to optimize receptor and arrestin construct expression. The similarity with rat Y1/βarr2 cell data is illustrated by agonist concentration–response curves in (A), for 60 min treatment with NPY (pEC50: 8.99 ± 0.08, n= 7), PYY (pEC50: 8.76 ± 0.09, n= 6), PP (n= 6), [Leu31, Pro34]NPY (ProNPY pEC50: 8.65 ± 0.06, n= 7) and NPY3–36 (n= 6). In (B), 30 min pre-incubation with 10 nM BIBO3304 (BIBO) resulted in a 9.1-fold rightward shift in the NPY concentration–response relationship (n= 5) from which a pKB of 8.9 was calculated.

Mentions: Stable expression of the human Y1 receptor is more difficult than for its rat orthologue, but we were also able to derive a dual transfected cell line capable of measuring the association of the human Y1 receptor with β-arrestin2 by BiFC ([125I]PYY Bmax: 610 ± 20 fmol·mg−1, n= 3). This demonstrated equivalent agonist and BIBO3304 pharmacology for both rat and human Y1 receptor/βarr2 association (Figure 7).


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)

Agonist and antagonist data for human Y1 receptor stimulated β-arrestin2 BiFC. Human hY1/βarr2 cells were derived after further dilution cloning to optimize receptor and arrestin construct expression. The similarity with rat Y1/βarr2 cell data is illustrated by agonist concentration–response curves in (A), for 60 min treatment with NPY (pEC50: 8.99 ± 0.08, n= 7), PYY (pEC50: 8.76 ± 0.09, n= 6), PP (n= 6), [Leu31, Pro34]NPY (ProNPY pEC50: 8.65 ± 0.06, n= 7) and NPY3–36 (n= 6). In (B), 30 min pre-incubation with 10 nM BIBO3304 (BIBO) resulted in a 9.1-fold rightward shift in the NPY concentration–response relationship (n= 5) from which a pKB of 8.9 was calculated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2901518&req=5

fig07: Agonist and antagonist data for human Y1 receptor stimulated β-arrestin2 BiFC. Human hY1/βarr2 cells were derived after further dilution cloning to optimize receptor and arrestin construct expression. The similarity with rat Y1/βarr2 cell data is illustrated by agonist concentration–response curves in (A), for 60 min treatment with NPY (pEC50: 8.99 ± 0.08, n= 7), PYY (pEC50: 8.76 ± 0.09, n= 6), PP (n= 6), [Leu31, Pro34]NPY (ProNPY pEC50: 8.65 ± 0.06, n= 7) and NPY3–36 (n= 6). In (B), 30 min pre-incubation with 10 nM BIBO3304 (BIBO) resulted in a 9.1-fold rightward shift in the NPY concentration–response relationship (n= 5) from which a pKB of 8.9 was calculated.
Mentions: Stable expression of the human Y1 receptor is more difficult than for its rat orthologue, but we were also able to derive a dual transfected cell line capable of measuring the association of the human Y1 receptor with β-arrestin2 by BiFC ([125I]PYY Bmax: 610 ± 20 fmol·mg−1, n= 3). This demonstrated equivalent agonist and BIBO3304 pharmacology for both rat and human Y1 receptor/βarr2 association (Figure 7).

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