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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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PAR2 facilitates interaction between PAR4 and 14-3-3ζ but disrupts interaction with β-COP1. PAR4 mECFP was transiently transfected into NCTC overexpressing PAR2 (NCTC-PAR2) cells. A, cells were treated, as described previously, to identify the plasma membrane (red) and nuclei (blue). Cells were visualized using a ×100 Plan Fluor objective. Images were merged to highlight distinct plasma membrane/nuclear compartments. Scale bars = 10 μm. Enhanced surface expression of PAR4 in NCTC-PAR2 cells is indicated by white arrows. B, protein expression was assessed using Western blotting in cells expressing increasing amounts of PAR4 mECFP with the protein bands were detected separated by subcellular fractionation in NCTC-PAR2 cells and resolved by Western blotting, as previously shown. C, enhanced surface expression was then quantitatively assessed by cell surface biotinylation of NCTC-2544 and NCTC-PAR2 cells expressing PAR4 mECFP. Interaction between (D) PAR4-HA and 14-3-3ζ or (E) PAR4-mECFP and the βCOP subunit of COPI was assessed by co-immunoprecipitation (IP) in NCTC-2544 and NCTC-PAR2 cells. Images and blots are representative of at least four independent experiments.
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Figure 4: PAR2 facilitates interaction between PAR4 and 14-3-3ζ but disrupts interaction with β-COP1. PAR4 mECFP was transiently transfected into NCTC overexpressing PAR2 (NCTC-PAR2) cells. A, cells were treated, as described previously, to identify the plasma membrane (red) and nuclei (blue). Cells were visualized using a ×100 Plan Fluor objective. Images were merged to highlight distinct plasma membrane/nuclear compartments. Scale bars = 10 μm. Enhanced surface expression of PAR4 in NCTC-PAR2 cells is indicated by white arrows. B, protein expression was assessed using Western blotting in cells expressing increasing amounts of PAR4 mECFP with the protein bands were detected separated by subcellular fractionation in NCTC-PAR2 cells and resolved by Western blotting, as previously shown. C, enhanced surface expression was then quantitatively assessed by cell surface biotinylation of NCTC-2544 and NCTC-PAR2 cells expressing PAR4 mECFP. Interaction between (D) PAR4-HA and 14-3-3ζ or (E) PAR4-mECFP and the βCOP subunit of COPI was assessed by co-immunoprecipitation (IP) in NCTC-2544 and NCTC-PAR2 cells. Images and blots are representative of at least four independent experiments.

Mentions: As shown in Fig. 4A, similar membrane translocation was observed for PAR4 mECFP when transfected into NCTC-PAR2. In addition, when PAR4 mECFP was resolved by Western blotting two clear protein forms were detected when expressed in NCTC-PAR2 cells (Fig. 4B). These results were similar to the observations made in previous experiments resolving the ER retention motif mutant PAR4 protein. Subsequent subcellular fractionation of NCTC-PAR2 cells expressing PAR4 mECFP highlighted the distinct differences in the compartmentalization of PAR4. As Fig. 4B shows, the more rapidly migrating 65-kDa species was confined to ER and endosomal compartments (lanes 4–6), whereas the distribution of the less rapidly migrating form strongly correlated with ER, endosomal, and plasma membrane fractions (lanes 1–6). Enhanced surface expression of PAR4 was subsequently quantified using cell surface biotinylation, as shown in Fig. 4C. Following biotinylation of surface proteins, expression of PAR4 was probed using both anti-GFP and anti-PAR4 specific antibodies in NCTC-2544 and NCTC-PAR2 cells transfected with PAR4 mECFP. Although detection of surface PAR4 was negligible in transfected NCTC-2544 cells (0.725 ± 0.30-fold increase over mock transfected cells, n = 4), a significant increase in surface PAR4 was detected in NCTC-PAR2-transfected cells (5.199 ± 0.85-fold increase over mock cells, n = 4). In addition to enhanced cell surface expression of PAR4 in the presence of PAR2, a notable increase in the ability of PAR4 to interact with the ζ isoform of the ER export chaperone 14-3-3 was detected in co-immunoprecipitation experiments (Fig. 4D). When PAR4-HA was expressed in the parental NCTC-2544 cells, the ability of 14-3-3ζ to interact with PAR4 was negligible. However, when PAR4-HA was expressed in NCTC-PAR2 cells, the ability of 14-3-3ζ to interact with PAR4 was clearly shown. Differential interaction of PAR4 with 14-3-3ζ was also demonstrated using GST pulldown assays employing GST-14-3-3ζ fusion proteins (supplemental Fig. S3). PAR4 binding to GST-14-3-3ζ was enhanced when expressed in NCTC-PAR2 cells. In addition, interaction between PAR4 and β-COP1 was no longer observed during co-expression of PAR2 and PAR4 (Fig. 4E and supplemental Fig. S3).


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)

PAR2 facilitates interaction between PAR4 and 14-3-3ζ but disrupts interaction with β-COP1. PAR4 mECFP was transiently transfected into NCTC overexpressing PAR2 (NCTC-PAR2) cells. A, cells were treated, as described previously, to identify the plasma membrane (red) and nuclei (blue). Cells were visualized using a ×100 Plan Fluor objective. Images were merged to highlight distinct plasma membrane/nuclear compartments. Scale bars = 10 μm. Enhanced surface expression of PAR4 in NCTC-PAR2 cells is indicated by white arrows. B, protein expression was assessed using Western blotting in cells expressing increasing amounts of PAR4 mECFP with the protein bands were detected separated by subcellular fractionation in NCTC-PAR2 cells and resolved by Western blotting, as previously shown. C, enhanced surface expression was then quantitatively assessed by cell surface biotinylation of NCTC-2544 and NCTC-PAR2 cells expressing PAR4 mECFP. Interaction between (D) PAR4-HA and 14-3-3ζ or (E) PAR4-mECFP and the βCOP subunit of COPI was assessed by co-immunoprecipitation (IP) in NCTC-2544 and NCTC-PAR2 cells. Images and blots are representative of at least four independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: PAR2 facilitates interaction between PAR4 and 14-3-3ζ but disrupts interaction with β-COP1. PAR4 mECFP was transiently transfected into NCTC overexpressing PAR2 (NCTC-PAR2) cells. A, cells were treated, as described previously, to identify the plasma membrane (red) and nuclei (blue). Cells were visualized using a ×100 Plan Fluor objective. Images were merged to highlight distinct plasma membrane/nuclear compartments. Scale bars = 10 μm. Enhanced surface expression of PAR4 in NCTC-PAR2 cells is indicated by white arrows. B, protein expression was assessed using Western blotting in cells expressing increasing amounts of PAR4 mECFP with the protein bands were detected separated by subcellular fractionation in NCTC-PAR2 cells and resolved by Western blotting, as previously shown. C, enhanced surface expression was then quantitatively assessed by cell surface biotinylation of NCTC-2544 and NCTC-PAR2 cells expressing PAR4 mECFP. Interaction between (D) PAR4-HA and 14-3-3ζ or (E) PAR4-mECFP and the βCOP subunit of COPI was assessed by co-immunoprecipitation (IP) in NCTC-2544 and NCTC-PAR2 cells. Images and blots are representative of at least four independent experiments.
Mentions: As shown in Fig. 4A, similar membrane translocation was observed for PAR4 mECFP when transfected into NCTC-PAR2. In addition, when PAR4 mECFP was resolved by Western blotting two clear protein forms were detected when expressed in NCTC-PAR2 cells (Fig. 4B). These results were similar to the observations made in previous experiments resolving the ER retention motif mutant PAR4 protein. Subsequent subcellular fractionation of NCTC-PAR2 cells expressing PAR4 mECFP highlighted the distinct differences in the compartmentalization of PAR4. As Fig. 4B shows, the more rapidly migrating 65-kDa species was confined to ER and endosomal compartments (lanes 4–6), whereas the distribution of the less rapidly migrating form strongly correlated with ER, endosomal, and plasma membrane fractions (lanes 1–6). Enhanced surface expression of PAR4 was subsequently quantified using cell surface biotinylation, as shown in Fig. 4C. Following biotinylation of surface proteins, expression of PAR4 was probed using both anti-GFP and anti-PAR4 specific antibodies in NCTC-2544 and NCTC-PAR2 cells transfected with PAR4 mECFP. Although detection of surface PAR4 was negligible in transfected NCTC-2544 cells (0.725 ± 0.30-fold increase over mock transfected cells, n = 4), a significant increase in surface PAR4 was detected in NCTC-PAR2-transfected cells (5.199 ± 0.85-fold increase over mock cells, n = 4). In addition to enhanced cell surface expression of PAR4 in the presence of PAR2, a notable increase in the ability of PAR4 to interact with the ζ isoform of the ER export chaperone 14-3-3 was detected in co-immunoprecipitation experiments (Fig. 4D). When PAR4-HA was expressed in the parental NCTC-2544 cells, the ability of 14-3-3ζ to interact with PAR4 was negligible. However, when PAR4-HA was expressed in NCTC-PAR2 cells, the ability of 14-3-3ζ to interact with PAR4 was clearly shown. Differential interaction of PAR4 with 14-3-3ζ was also demonstrated using GST pulldown assays employing GST-14-3-3ζ fusion proteins (supplemental Fig. S3). PAR4 binding to GST-14-3-3ζ was enhanced when expressed in NCTC-PAR2 cells. In addition, interaction between PAR4 and β-COP1 was no longer observed during co-expression of PAR2 and PAR4 (Fig. 4E and supplemental Fig. S3).

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
Related in: MedlinePlus