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Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4).

Cunningham MR, McIntosh KA, Pediani JD, Robben J, Cooke AE, Nilsson M, Gould GW, Mundell S, Milligan G, Plevin R - J. Biol. Chem. (2012)

Bottom Line: Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ.Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4).Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4).

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, Univesity of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, Scotland, United Kingdom. margaret.cunningham@bristol.ac.uk

ABSTRACT
Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR → A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.

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FRET imaging and co-immunoprecipitation reveals heterodimer formation between PAR2 and PAR4 in HEK293 cells. PAR4 mECFP and PAR2 mEYFP were co-expressed in HEK293 cells. Wide field FRET imaging was performed in live cells. A, images were acquired for CFP, YFP, uncorrected FRET (uFRET), with the uFRET channel corrected for spectral bleed-through/contamination (cFRET). Scale bars = 25 μm. Corresponding ratiometric FRET values were then quantified and graphed. Data are expressed as mean ± S.E. from three separate FRET experiments (n = 72 single cell measurements), **, p = 0.001 one-way analysis of variance with Dunnett's post-test. B, interaction between PAR4-HA and PAR2 mEYFP was further assessed by co-immunoprecipitation (IP). Blots are representative of at least three independent experiments.
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Figure 6: FRET imaging and co-immunoprecipitation reveals heterodimer formation between PAR2 and PAR4 in HEK293 cells. PAR4 mECFP and PAR2 mEYFP were co-expressed in HEK293 cells. Wide field FRET imaging was performed in live cells. A, images were acquired for CFP, YFP, uncorrected FRET (uFRET), with the uFRET channel corrected for spectral bleed-through/contamination (cFRET). Scale bars = 25 μm. Corresponding ratiometric FRET values were then quantified and graphed. Data are expressed as mean ± S.E. from three separate FRET experiments (n = 72 single cell measurements), **, p = 0.001 one-way analysis of variance with Dunnett's post-test. B, interaction between PAR4-HA and PAR2 mEYFP was further assessed by co-immunoprecipitation (IP). Blots are representative of at least three independent experiments.

Mentions: The novel features of PAR2/PAR4 co-expression were investigated further to identify if enhancement of the PAR4 cell surface expression was a result of interaction between PAR2 and PAR4. For this purpose, wide field intermolecular FRET imaging was carried out (41, 42) in HEK293 cells expressing either PAR4 mECFP or PAR2 mEYFP alone or co-expressing these two constructs. As shown in Fig. 6A, an intracellular FRET signal was observed, presumably in the ER and/or Golgi complex, with a weak signal observed at the plasma membrane. When quantified, co-expression of PAR2 mEYFP and PAR4 mECFP resulted in a significant increase in RFRET (1.883 ± 0.003) when compared with experimental conditions where collisional FRET could occur, i.e. co-expression of mEYFP and mECFP in cells yielded a RFRET value of 1.173 ± 0.055. Interaction between PAR2 and PAR4 was also demonstrated by co-immunoprecipitation in HEK293 cells as shown in Fig. 6B. These data indicate that PAR2/PAR4 heterodimerization occurs and is likely responsible for enhanced cell surface expression of PAR4.


Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4).

Cunningham MR, McIntosh KA, Pediani JD, Robben J, Cooke AE, Nilsson M, Gould GW, Mundell S, Milligan G, Plevin R - J. Biol. Chem. (2012)

FRET imaging and co-immunoprecipitation reveals heterodimer formation between PAR2 and PAR4 in HEK293 cells. PAR4 mECFP and PAR2 mEYFP were co-expressed in HEK293 cells. Wide field FRET imaging was performed in live cells. A, images were acquired for CFP, YFP, uncorrected FRET (uFRET), with the uFRET channel corrected for spectral bleed-through/contamination (cFRET). Scale bars = 25 μm. Corresponding ratiometric FRET values were then quantified and graphed. Data are expressed as mean ± S.E. from three separate FRET experiments (n = 72 single cell measurements), **, p = 0.001 one-way analysis of variance with Dunnett's post-test. B, interaction between PAR4-HA and PAR2 mEYFP was further assessed by co-immunoprecipitation (IP). Blots are representative of at least three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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Figure 6: FRET imaging and co-immunoprecipitation reveals heterodimer formation between PAR2 and PAR4 in HEK293 cells. PAR4 mECFP and PAR2 mEYFP were co-expressed in HEK293 cells. Wide field FRET imaging was performed in live cells. A, images were acquired for CFP, YFP, uncorrected FRET (uFRET), with the uFRET channel corrected for spectral bleed-through/contamination (cFRET). Scale bars = 25 μm. Corresponding ratiometric FRET values were then quantified and graphed. Data are expressed as mean ± S.E. from three separate FRET experiments (n = 72 single cell measurements), **, p = 0.001 one-way analysis of variance with Dunnett's post-test. B, interaction between PAR4-HA and PAR2 mEYFP was further assessed by co-immunoprecipitation (IP). Blots are representative of at least three independent experiments.
Mentions: The novel features of PAR2/PAR4 co-expression were investigated further to identify if enhancement of the PAR4 cell surface expression was a result of interaction between PAR2 and PAR4. For this purpose, wide field intermolecular FRET imaging was carried out (41, 42) in HEK293 cells expressing either PAR4 mECFP or PAR2 mEYFP alone or co-expressing these two constructs. As shown in Fig. 6A, an intracellular FRET signal was observed, presumably in the ER and/or Golgi complex, with a weak signal observed at the plasma membrane. When quantified, co-expression of PAR2 mEYFP and PAR4 mECFP resulted in a significant increase in RFRET (1.883 ± 0.003) when compared with experimental conditions where collisional FRET could occur, i.e. co-expression of mEYFP and mECFP in cells yielded a RFRET value of 1.173 ± 0.055. Interaction between PAR2 and PAR4 was also demonstrated by co-immunoprecipitation in HEK293 cells as shown in Fig. 6B. These data indicate that PAR2/PAR4 heterodimerization occurs and is likely responsible for enhanced cell surface expression of PAR4.

Bottom Line: Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ.Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4).Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4).

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, Univesity of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, Scotland, United Kingdom. margaret.cunningham@bristol.ac.uk

ABSTRACT
Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR → A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.

Show MeSH