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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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Mutating Y1 receptor C tail phosphorylation sites inhibits β-arrestin2 association. The upper diagram shows the C-terminal amino acid sequence of the rat Y1 receptor, highlighting the region between positions 352 and 362, which contains six Ser/Thr residues (white text on green) and which was identified previously as the key phosphorylated sequence in the receptor (Holliday et al., 2005). These Ser/Thr residues were mutated individually or in combination, and eight stable Y1 mutant/βarr2 cell lines established with closely matched expression levels to Y1/βarr2 cells (Table 2). NPY- and PYY-stimulated β-arrestin2 BiFC responses were measured for each mutant and normalized by comparison with basal and 1 µM NPY Y1/βarr2 control responses on the same plate. Each graph presents the pooled data (n= 4–5) with the location of the Ala substitutions (white text on red) highlighted in the diagram above (STA = S352A, T353A; TSA = T361A, S362A; 5A = S352A, T353A, S359A, T361A, S362A; 6A = S352A, T353A, T356A, S359A, T361A, S362A). In each case, the dotted curve represents the NPY concentration–response curve for Y1/βarr2 cells (as Figure 5B). The curve fits yielded the pEC50 estimates in Table 3.
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fig08: Mutating Y1 receptor C tail phosphorylation sites inhibits β-arrestin2 association. The upper diagram shows the C-terminal amino acid sequence of the rat Y1 receptor, highlighting the region between positions 352 and 362, which contains six Ser/Thr residues (white text on green) and which was identified previously as the key phosphorylated sequence in the receptor (Holliday et al., 2005). These Ser/Thr residues were mutated individually or in combination, and eight stable Y1 mutant/βarr2 cell lines established with closely matched expression levels to Y1/βarr2 cells (Table 2). NPY- and PYY-stimulated β-arrestin2 BiFC responses were measured for each mutant and normalized by comparison with basal and 1 µM NPY Y1/βarr2 control responses on the same plate. Each graph presents the pooled data (n= 4–5) with the location of the Ala substitutions (white text on red) highlighted in the diagram above (STA = S352A, T353A; TSA = T361A, S362A; 5A = S352A, T353A, S359A, T361A, S362A; 6A = S352A, T353A, T356A, S359A, T361A, S362A). In each case, the dotted curve represents the NPY concentration–response curve for Y1/βarr2 cells (as Figure 5B). The curve fits yielded the pEC50 estimates in Table 3.

Mentions: Data are obtained from 4–10 experiments (see also concentration–response curves in Figures 8 and 10). pEC50s are not quoted (–) in mutants where agonist responses were too small for accurate estimates. Some mutants also changed basal β-arrestin2 BiFC as shown in Figures 8 and 10.


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)

Mutating Y1 receptor C tail phosphorylation sites inhibits β-arrestin2 association. The upper diagram shows the C-terminal amino acid sequence of the rat Y1 receptor, highlighting the region between positions 352 and 362, which contains six Ser/Thr residues (white text on green) and which was identified previously as the key phosphorylated sequence in the receptor (Holliday et al., 2005). These Ser/Thr residues were mutated individually or in combination, and eight stable Y1 mutant/βarr2 cell lines established with closely matched expression levels to Y1/βarr2 cells (Table 2). NPY- and PYY-stimulated β-arrestin2 BiFC responses were measured for each mutant and normalized by comparison with basal and 1 µM NPY Y1/βarr2 control responses on the same plate. Each graph presents the pooled data (n= 4–5) with the location of the Ala substitutions (white text on red) highlighted in the diagram above (STA = S352A, T353A; TSA = T361A, S362A; 5A = S352A, T353A, S359A, T361A, S362A; 6A = S352A, T353A, T356A, S359A, T361A, S362A). In each case, the dotted curve represents the NPY concentration–response curve for Y1/βarr2 cells (as Figure 5B). The curve fits yielded the pEC50 estimates in Table 3.
© Copyright Policy
Related In: Results  -  Collection

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

fig08: Mutating Y1 receptor C tail phosphorylation sites inhibits β-arrestin2 association. The upper diagram shows the C-terminal amino acid sequence of the rat Y1 receptor, highlighting the region between positions 352 and 362, which contains six Ser/Thr residues (white text on green) and which was identified previously as the key phosphorylated sequence in the receptor (Holliday et al., 2005). These Ser/Thr residues were mutated individually or in combination, and eight stable Y1 mutant/βarr2 cell lines established with closely matched expression levels to Y1/βarr2 cells (Table 2). NPY- and PYY-stimulated β-arrestin2 BiFC responses were measured for each mutant and normalized by comparison with basal and 1 µM NPY Y1/βarr2 control responses on the same plate. Each graph presents the pooled data (n= 4–5) with the location of the Ala substitutions (white text on red) highlighted in the diagram above (STA = S352A, T353A; TSA = T361A, S362A; 5A = S352A, T353A, S359A, T361A, S362A; 6A = S352A, T353A, T356A, S359A, T361A, S362A). In each case, the dotted curve represents the NPY concentration–response curve for Y1/βarr2 cells (as Figure 5B). The curve fits yielded the pEC50 estimates in Table 3.
Mentions: Data are obtained from 4–10 experiments (see also concentration–response curves in Figures 8 and 10). pEC50s are not quoted (–) in mutants where agonist responses were too small for accurate estimates. Some mutants also changed basal β-arrestin2 BiFC as shown in Figures 8 and 10.

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