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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
Principle of the 7TMR–β-arrestin2 BiFC assay. Reversible association of β-arrestin2–Yn with NPY-stimulated Y1–Yc receptors promotes stable Yn and Yc fragment refolding. The formation of the YFP β-barrel allows a slower oxidation reaction to generate the internal chromophore, resulting in cYFP fluorescence as an indicator of the interaction.
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fig01: Principle of the 7TMR–β-arrestin2 BiFC assay. Reversible association of β-arrestin2–Yn with NPY-stimulated Y1–Yc receptors promotes stable Yn and Yc fragment refolding. The formation of the YFP β-barrel allows a slower oxidation reaction to generate the internal chromophore, resulting in cYFP fluorescence as an indicator of the interaction.

Mentions: Bimolecular fluorescence complementation (BiFC) is an alternative technique for detecting protein–protein association in live cells (Kerppola, 2008; Rose et al., 2010). The investigated proteins are tagged with split N- and C-terminal fragments of an Aequorea victoria-based fluorescent protein, such as the yellow variant (YFP). These fragments (Yn, Yc) are themselves non-fluorescent, but interaction between the tagged proteins brings Yn and Yc together and promotes their refolding into the YFP β-barrel structure. Natural maturation of the chromophore then regenerates YFP fluorescence, giving a simple single-wavelength signal indicating protein–protein association (Figure 1). BiFC can be localized at a subcellular level, and a wide intensity range enhances sensitivity (Kerppola, 2008). Compromises include the slow maturation of complemented YFP (cYFP), which delays the onset of the BiFC response, and BiFC complexes also appear irreversible once formed (Hu et al., 2002). Perhaps because of this, many BiFC studies have focused on stable interactions, for example, between transcription factor dimers (Hu et al., 2002), Gβγ subunits (Mervine et al., 2006) or 7TMR dimers (Briddon et al., 2008; Gandia et al., 2008; Vidi et al., 2008). However, the first fragment refolding step commits a particular interaction to form a BiFC complex, and this is fast enough (half-time of 60 s in vitro; Hu et al., 2002) to trap more transient associations typical of signalling proteins (Morell et al., 2007). In the most complete illustration of this possibility, a range of BiFC-based reporters quantified the concerted actions of different compounds on various signalling, mitogenic and apoptotic pathways (MacDonald et al., 2006). This study also confirmed that in principle, the recruitment of β-arrestin2 to the β2-adrenoceptor could be detected by BiFC reporters (Auld et al., 2006; MacDonald et al., 2006).


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)

Principle of the 7TMR–β-arrestin2 BiFC assay. Reversible association of β-arrestin2–Yn with NPY-stimulated Y1–Yc receptors promotes stable Yn and Yc fragment refolding. The formation of the YFP β-barrel allows a slower oxidation reaction to generate the internal chromophore, resulting in cYFP fluorescence as an indicator of the interaction.
© Copyright Policy
Related In: Results  -  Collection

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

fig01: Principle of the 7TMR–β-arrestin2 BiFC assay. Reversible association of β-arrestin2–Yn with NPY-stimulated Y1–Yc receptors promotes stable Yn and Yc fragment refolding. The formation of the YFP β-barrel allows a slower oxidation reaction to generate the internal chromophore, resulting in cYFP fluorescence as an indicator of the interaction.
Mentions: Bimolecular fluorescence complementation (BiFC) is an alternative technique for detecting protein–protein association in live cells (Kerppola, 2008; Rose et al., 2010). The investigated proteins are tagged with split N- and C-terminal fragments of an Aequorea victoria-based fluorescent protein, such as the yellow variant (YFP). These fragments (Yn, Yc) are themselves non-fluorescent, but interaction between the tagged proteins brings Yn and Yc together and promotes their refolding into the YFP β-barrel structure. Natural maturation of the chromophore then regenerates YFP fluorescence, giving a simple single-wavelength signal indicating protein–protein association (Figure 1). BiFC can be localized at a subcellular level, and a wide intensity range enhances sensitivity (Kerppola, 2008). Compromises include the slow maturation of complemented YFP (cYFP), which delays the onset of the BiFC response, and BiFC complexes also appear irreversible once formed (Hu et al., 2002). Perhaps because of this, many BiFC studies have focused on stable interactions, for example, between transcription factor dimers (Hu et al., 2002), Gβγ subunits (Mervine et al., 2006) or 7TMR dimers (Briddon et al., 2008; Gandia et al., 2008; Vidi et al., 2008). However, the first fragment refolding step commits a particular interaction to form a BiFC complex, and this is fast enough (half-time of 60 s in vitro; Hu et al., 2002) to trap more transient associations typical of signalling proteins (Morell et al., 2007). In the most complete illustration of this possibility, a range of BiFC-based reporters quantified the concerted actions of different compounds on various signalling, mitogenic and apoptotic pathways (MacDonald et al., 2006). This study also confirmed that in principle, the recruitment of β-arrestin2 to the β2-adrenoceptor could be detected by BiFC reporters (Auld et al., 2006; MacDonald et al., 2006).

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