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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
NPY specifically stimulates Y1 receptor–β-arrestin2 BiFC. HEK293 cells were transiently co-transfected with FLAG-tagged Y1 receptor–Yc and β-arrestin–Yn plasmids. Cells were live labelled 24 h later with M2 anti-FLAG antibody before treatment with vehicle or 1 µM NPY for 60 min at 37°C. Following fixation, the M2 antibody was identified in fixed cells by a secondary antibody conjugated to Rhodamine Red X. Images were acquired using a Zeiss LSM510 confocal microscope with laser excitation at 488 nm (cYFP BiFC fluorescence, and phase) or 543 nm (M2 detected FLAG Y1 receptors). Representative images are shown from one of at least three experiments. They illustrate the increase in BiFC fluorescence, co-localized with receptor immunoreactivity, in NPY-stimulated cells co-transfected with Y1–Yc and β-arrestin2–Yn. This response was absent when using either a Y1 receptor–Yc construct lacking the C tail after Tyr346 (ΔY346), or a dominant negative (DN) truncated β-arrestin1(319–418)–Yn.
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fig02: NPY specifically stimulates Y1 receptor–β-arrestin2 BiFC. HEK293 cells were transiently co-transfected with FLAG-tagged Y1 receptor–Yc and β-arrestin–Yn plasmids. Cells were live labelled 24 h later with M2 anti-FLAG antibody before treatment with vehicle or 1 µM NPY for 60 min at 37°C. Following fixation, the M2 antibody was identified in fixed cells by a secondary antibody conjugated to Rhodamine Red X. Images were acquired using a Zeiss LSM510 confocal microscope with laser excitation at 488 nm (cYFP BiFC fluorescence, and phase) or 543 nm (M2 detected FLAG Y1 receptors). Representative images are shown from one of at least three experiments. They illustrate the increase in BiFC fluorescence, co-localized with receptor immunoreactivity, in NPY-stimulated cells co-transfected with Y1–Yc and β-arrestin2–Yn. This response was absent when using either a Y1 receptor–Yc construct lacking the C tail after Tyr346 (ΔY346), or a dominant negative (DN) truncated β-arrestin1(319–418)–Yn.

Mentions: We used BiFC fragment pairs based on a rapid folding YFP variant called venus (Nagai et al., 2002), to maximize trapping of agonist-induced Y1 receptor–β-arrestin2 complexes at physiological temperature (37°C). Early optimization suggested that C-terminal tagging of the rat Y1 receptor with Yc (155–238) was most suitable, in combination with β-arrestin2 fused at the C-terminus to Yn (1–173). This overlapping pair (N173:C155) repeats a β-strand (155–172) in both Yn and Yc, as an aid to the refolding process (Hu and Kerppola, 2003). Y1–Yc and βarr2–Yn cDNAs were co-transfected into HEK293 cells, and receptors were identified by live antibody labelling of the N-terminal FLAG epitope (Figure 2). Under control conditions, relatively low levels of cYFP fluorescence were observed, but following 60 min treatment with NPY, there was a marked increase in BiFC. This was located principally in perinuclear intracellular compartments and largely co-localized with receptor immunoreactivity. In contrast, the agonist-induced BiFC response was absent when using a truncated Y1 receptor construct lacking much of the C-terminal tail after the palmitoylation site (ΔY346), or when full-length Y1–Yc was partnered with truncated β-arr1(318–419)–Yn without receptor binding domains (Figure 2). In both these controls, Y1 receptor internalization was also largely prevented.


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)

NPY specifically stimulates Y1 receptor–β-arrestin2 BiFC. HEK293 cells were transiently co-transfected with FLAG-tagged Y1 receptor–Yc and β-arrestin–Yn plasmids. Cells were live labelled 24 h later with M2 anti-FLAG antibody before treatment with vehicle or 1 µM NPY for 60 min at 37°C. Following fixation, the M2 antibody was identified in fixed cells by a secondary antibody conjugated to Rhodamine Red X. Images were acquired using a Zeiss LSM510 confocal microscope with laser excitation at 488 nm (cYFP BiFC fluorescence, and phase) or 543 nm (M2 detected FLAG Y1 receptors). Representative images are shown from one of at least three experiments. They illustrate the increase in BiFC fluorescence, co-localized with receptor immunoreactivity, in NPY-stimulated cells co-transfected with Y1–Yc and β-arrestin2–Yn. This response was absent when using either a Y1 receptor–Yc construct lacking the C tail after Tyr346 (ΔY346), or a dominant negative (DN) truncated β-arrestin1(319–418)–Yn.
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Related In: Results  -  Collection

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fig02: NPY specifically stimulates Y1 receptor–β-arrestin2 BiFC. HEK293 cells were transiently co-transfected with FLAG-tagged Y1 receptor–Yc and β-arrestin–Yn plasmids. Cells were live labelled 24 h later with M2 anti-FLAG antibody before treatment with vehicle or 1 µM NPY for 60 min at 37°C. Following fixation, the M2 antibody was identified in fixed cells by a secondary antibody conjugated to Rhodamine Red X. Images were acquired using a Zeiss LSM510 confocal microscope with laser excitation at 488 nm (cYFP BiFC fluorescence, and phase) or 543 nm (M2 detected FLAG Y1 receptors). Representative images are shown from one of at least three experiments. They illustrate the increase in BiFC fluorescence, co-localized with receptor immunoreactivity, in NPY-stimulated cells co-transfected with Y1–Yc and β-arrestin2–Yn. This response was absent when using either a Y1 receptor–Yc construct lacking the C tail after Tyr346 (ΔY346), or a dominant negative (DN) truncated β-arrestin1(319–418)–Yn.
Mentions: We used BiFC fragment pairs based on a rapid folding YFP variant called venus (Nagai et al., 2002), to maximize trapping of agonist-induced Y1 receptor–β-arrestin2 complexes at physiological temperature (37°C). Early optimization suggested that C-terminal tagging of the rat Y1 receptor with Yc (155–238) was most suitable, in combination with β-arrestin2 fused at the C-terminus to Yn (1–173). This overlapping pair (N173:C155) repeats a β-strand (155–172) in both Yn and Yc, as an aid to the refolding process (Hu and Kerppola, 2003). Y1–Yc and βarr2–Yn cDNAs were co-transfected into HEK293 cells, and receptors were identified by live antibody labelling of the N-terminal FLAG epitope (Figure 2). Under control conditions, relatively low levels of cYFP fluorescence were observed, but following 60 min treatment with NPY, there was a marked increase in BiFC. This was located principally in perinuclear intracellular compartments and largely co-localized with receptor immunoreactivity. In contrast, the agonist-induced BiFC response was absent when using a truncated Y1 receptor construct lacking much of the C-terminal tail after the palmitoylation site (ΔY346), or when full-length Y1–Yc was partnered with truncated β-arr1(318–419)–Yn without receptor binding domains (Figure 2). In both these controls, Y1 receptor internalization was also largely prevented.

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