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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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Mutation of the arginine-based ER retention motif (R183AR) enhances the cell surface expression of PAR4.A, NCTC-2544 cells expressing PAR4 mECFP or PAR4 RAR mECFP mutant (green) were treated to stain for the plasma membrane (PM, red) and nucleus (blue) as previously described. Cells were visualized using a ×100 Plan Fluor objective. Scale bars = 10 μm. Intracellular retention of PAR4 mECFP is highlighted (red arrows), whereas notable membrane localization of the PAR4 RAR mECFP is evident (white arrows). B, protein expression was assessed using Western blotting of whole cell lysates expressing increasing amounts of PAR4 mECFP or PAR4 RAR mECFP constructs as indicated. C, changes in the surface expression of PAR4 were confirmed by subcellular fractionation using differential ultracentrifugation on an iodixanol gradient in cells expressing PAR4 mECFP or PAR4 RAR mECFP. Fractions were precipitated and resolved by SDS-PAGE followed by Western blotting. PAR4 mECFP (predicted band size ∼65 kDa) was detected using a polyclonal GFP antibody capable of recognizing the ECFP at the C-terminal of PAR4. Na+,K+-ATPase (∼100 kDa), transferrin receptor (∼190 kDa), and calnexin (90 ∼kDa) antibodies were used for the detection of membrane, endosomal, and ER compartments, respectively. D, interaction between PAR4 and COPI complex proteins was confirmed by co-immunoprecipitation. Lysates from NCTC-2544 cells expressing PAR4 mECFP were subjected to immunoprecipitation (IP) and then probed for β-COP1 interaction. Images and blots are representative of three separate experiments.
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Figure 2: Mutation of the arginine-based ER retention motif (R183AR) enhances the cell surface expression of PAR4.A, NCTC-2544 cells expressing PAR4 mECFP or PAR4 RAR mECFP mutant (green) were treated to stain for the plasma membrane (PM, red) and nucleus (blue) as previously described. Cells were visualized using a ×100 Plan Fluor objective. Scale bars = 10 μm. Intracellular retention of PAR4 mECFP is highlighted (red arrows), whereas notable membrane localization of the PAR4 RAR mECFP is evident (white arrows). B, protein expression was assessed using Western blotting of whole cell lysates expressing increasing amounts of PAR4 mECFP or PAR4 RAR mECFP constructs as indicated. C, changes in the surface expression of PAR4 were confirmed by subcellular fractionation using differential ultracentrifugation on an iodixanol gradient in cells expressing PAR4 mECFP or PAR4 RAR mECFP. Fractions were precipitated and resolved by SDS-PAGE followed by Western blotting. PAR4 mECFP (predicted band size ∼65 kDa) was detected using a polyclonal GFP antibody capable of recognizing the ECFP at the C-terminal of PAR4. Na+,K+-ATPase (∼100 kDa), transferrin receptor (∼190 kDa), and calnexin (90 ∼kDa) antibodies were used for the detection of membrane, endosomal, and ER compartments, respectively. D, interaction between PAR4 and COPI complex proteins was confirmed by co-immunoprecipitation. Lysates from NCTC-2544 cells expressing PAR4 mECFP were subjected to immunoprecipitation (IP) and then probed for β-COP1 interaction. Images and blots are representative of three separate experiments.

Mentions: Analysis of the protein sequence for PAR4 identified two potential arginine-based (RXR) ER retention motifs located within the intracellular loop-2 of the receptor (supplemental Fig. S1). Alignment of the primary sequences for all PAR family members found that these motifs were unique to PAR4. The contribution of these motifs in controlling the cellular localization of PAR4 was assessed by removing the arginine residues by alanine substitution (RXR → AXA). Of the possible motifs investigated, only mutation of the R183AR to A183AA resulted in a loss of ER retention and allowed PAR4 to translocate to the plasma membrane (Fig. 2A). Receptor expression levels were determined by Western blotting (Fig. 2B). Following expression of PAR4 mECFP the appearance of a protein band, resolving around 65 kDa, was observed. This corresponded well with the predicted molecular mass of PAR4 mECFP (38 kDa for PAR4 combined with 27 kDa for the mECFP). As Fig. 2B shows, as the expression of the R183AR mutant increased, the appearance of multiple protein forms was observed, a doublet resolving around 65 kDa and a slightly larger species resolving between 70 and 80 kDa. Subcellular fractionation of cells expressing either PAR4 mECFP or the R183AR mutant was carried out to separate plasma membrane, endosomal, and ER compartments followed by Western blot (Fig. 2C). The 65-kDa protein species observed in cells expressing PAR4 mECFP or mutant receptor reached maximal levels in ER and endosomal fractions (lanes 4–7), co-locating with calnexin and transferrin markers, respectively. These experiments identified that the higher molecular mass species observed in cells expressing the R183AR mutant reflected receptors located in the plasma membrane and endosome compartments (lanes 1–4) as shown using Na+,K+-ATPase and transferrin receptor markers, respectively.


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)

Mutation of the arginine-based ER retention motif (R183AR) enhances the cell surface expression of PAR4.A, NCTC-2544 cells expressing PAR4 mECFP or PAR4 RAR mECFP mutant (green) were treated to stain for the plasma membrane (PM, red) and nucleus (blue) as previously described. Cells were visualized using a ×100 Plan Fluor objective. Scale bars = 10 μm. Intracellular retention of PAR4 mECFP is highlighted (red arrows), whereas notable membrane localization of the PAR4 RAR mECFP is evident (white arrows). B, protein expression was assessed using Western blotting of whole cell lysates expressing increasing amounts of PAR4 mECFP or PAR4 RAR mECFP constructs as indicated. C, changes in the surface expression of PAR4 were confirmed by subcellular fractionation using differential ultracentrifugation on an iodixanol gradient in cells expressing PAR4 mECFP or PAR4 RAR mECFP. Fractions were precipitated and resolved by SDS-PAGE followed by Western blotting. PAR4 mECFP (predicted band size ∼65 kDa) was detected using a polyclonal GFP antibody capable of recognizing the ECFP at the C-terminal of PAR4. Na+,K+-ATPase (∼100 kDa), transferrin receptor (∼190 kDa), and calnexin (90 ∼kDa) antibodies were used for the detection of membrane, endosomal, and ER compartments, respectively. D, interaction between PAR4 and COPI complex proteins was confirmed by co-immunoprecipitation. Lysates from NCTC-2544 cells expressing PAR4 mECFP were subjected to immunoprecipitation (IP) and then probed for β-COP1 interaction. Images and blots are representative of three separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Mutation of the arginine-based ER retention motif (R183AR) enhances the cell surface expression of PAR4.A, NCTC-2544 cells expressing PAR4 mECFP or PAR4 RAR mECFP mutant (green) were treated to stain for the plasma membrane (PM, red) and nucleus (blue) as previously described. Cells were visualized using a ×100 Plan Fluor objective. Scale bars = 10 μm. Intracellular retention of PAR4 mECFP is highlighted (red arrows), whereas notable membrane localization of the PAR4 RAR mECFP is evident (white arrows). B, protein expression was assessed using Western blotting of whole cell lysates expressing increasing amounts of PAR4 mECFP or PAR4 RAR mECFP constructs as indicated. C, changes in the surface expression of PAR4 were confirmed by subcellular fractionation using differential ultracentrifugation on an iodixanol gradient in cells expressing PAR4 mECFP or PAR4 RAR mECFP. Fractions were precipitated and resolved by SDS-PAGE followed by Western blotting. PAR4 mECFP (predicted band size ∼65 kDa) was detected using a polyclonal GFP antibody capable of recognizing the ECFP at the C-terminal of PAR4. Na+,K+-ATPase (∼100 kDa), transferrin receptor (∼190 kDa), and calnexin (90 ∼kDa) antibodies were used for the detection of membrane, endosomal, and ER compartments, respectively. D, interaction between PAR4 and COPI complex proteins was confirmed by co-immunoprecipitation. Lysates from NCTC-2544 cells expressing PAR4 mECFP were subjected to immunoprecipitation (IP) and then probed for β-COP1 interaction. Images and blots are representative of three separate experiments.
Mentions: Analysis of the protein sequence for PAR4 identified two potential arginine-based (RXR) ER retention motifs located within the intracellular loop-2 of the receptor (supplemental Fig. S1). Alignment of the primary sequences for all PAR family members found that these motifs were unique to PAR4. The contribution of these motifs in controlling the cellular localization of PAR4 was assessed by removing the arginine residues by alanine substitution (RXR → AXA). Of the possible motifs investigated, only mutation of the R183AR to A183AA resulted in a loss of ER retention and allowed PAR4 to translocate to the plasma membrane (Fig. 2A). Receptor expression levels were determined by Western blotting (Fig. 2B). Following expression of PAR4 mECFP the appearance of a protein band, resolving around 65 kDa, was observed. This corresponded well with the predicted molecular mass of PAR4 mECFP (38 kDa for PAR4 combined with 27 kDa for the mECFP). As Fig. 2B shows, as the expression of the R183AR mutant increased, the appearance of multiple protein forms was observed, a doublet resolving around 65 kDa and a slightly larger species resolving between 70 and 80 kDa. Subcellular fractionation of cells expressing either PAR4 mECFP or the R183AR mutant was carried out to separate plasma membrane, endosomal, and ER compartments followed by Western blot (Fig. 2C). The 65-kDa protein species observed in cells expressing PAR4 mECFP or mutant receptor reached maximal levels in ER and endosomal fractions (lanes 4–7), co-locating with calnexin and transferrin markers, respectively. These experiments identified that the higher molecular mass species observed in cells expressing the R183AR mutant reflected receptors located in the plasma membrane and endosome compartments (lanes 1–4) as shown using Na+,K+-ATPase and transferrin receptor markers, respectively.

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