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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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The presence of PAR2 allows N-linked glycosylation of PAR4, which promotes membrane localization. HEK293 cells were transfected with (A) PAR4 mECFP or PAR4 RAR mutant constructs. Prevention of de novo N-glycosylation was carried out by the addition of tunicamycin (2 μg/ml) to transfected cells as indicated for 16 h. B, NCTC, NCTC-PAR2, and HEK293 cells were transfected with increasing concentrations of the PAR4 N56A mECFP mutant or PAR4 mECFP. Whole cell lysates were prepared and resolved by Western blotting. C, fluorescent microscopy confirmed a loss of surface localization of PAR4 following mutation of N-glycosylation site (red arrows). Cells were visualized using a ×100 Plan Fluor objective. Scale bars = 10 μm. Blots and images are representative of three independent experiments.
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Figure 7: The presence of PAR2 allows N-linked glycosylation of PAR4, which promotes membrane localization. HEK293 cells were transfected with (A) PAR4 mECFP or PAR4 RAR mutant constructs. Prevention of de novo N-glycosylation was carried out by the addition of tunicamycin (2 μg/ml) to transfected cells as indicated for 16 h. B, NCTC, NCTC-PAR2, and HEK293 cells were transfected with increasing concentrations of the PAR4 N56A mECFP mutant or PAR4 mECFP. Whole cell lysates were prepared and resolved by Western blotting. C, fluorescent microscopy confirmed a loss of surface localization of PAR4 following mutation of N-glycosylation site (red arrows). Cells were visualized using a ×100 Plan Fluor objective. Scale bars = 10 μm. Blots and images are representative of three independent experiments.

Mentions: We then examined the role of PAR2 in regulating further post-translational modification of PAR4 in the context of receptor maturation and cell surface expression. N-Linked complex glycosylation is an important post-translational modification for efficient cell surface delivery of GPCRs (46). Analysis of the protein sequence of PAR4 identified an Asn-Xxx-serine/threonine N-glycosylation motif located at position 56 (N56DS) in the N terminus of the receptor (supplemental Fig. S1), suggesting that PAR4 may undergo N-linked glycosylation. To assess this, first, pharmacological inhibition of glycosylation was carried out using the GlcNAc phosphotransferase inhibitor tunicamycin, which prevents all N-linked glycosylation. Fig. 7A shows that pretreatment of HEK293 cells with tunicamycin eliminated the higher molecular mass form of PAR4, whereas the lower 65-kDa protein was still retained, with a minor intermediate band indicated. This effect was replicated in the R183AR to A183AA mutant, known to be preferentially expressed at the membrane. Experiments were then conducted using an N-terminal mutant of PAR4 (Asn56 → Ala56) to determine the effect of PAR2 upon protein species expression (Fig. 7B) and receptor localization (Fig. 7C). In control NCTC cells the PAR4 N56A mutant construct (Fig. 7B, top panel) was expressed as a single 65-kDa protein form that corresponded to wild type PAR4 mECFP. When expressed in NCTC-PAR2 cells (Fig. 7B, middle panel) a loss in the higher molecular mass protein form was observed in comparison to wild type PAR4 mECFP, however, the 65-kDa species was expressed alongside the slightly higher molecular mass form previously observed in the tunicamycin inhibition experiments. Similar results were obtained following expression in HEK293 cells (Fig. 7B, lower panel). Corresponding fluorescence microscopy images are shown in Fig. 7C. Lack of PAR4 cell surface expression was observed following expression of the PAR4 N56A mutant in NCTC-PAR2 cells.


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)

The presence of PAR2 allows N-linked glycosylation of PAR4, which promotes membrane localization. HEK293 cells were transfected with (A) PAR4 mECFP or PAR4 RAR mutant constructs. Prevention of de novo N-glycosylation was carried out by the addition of tunicamycin (2 μg/ml) to transfected cells as indicated for 16 h. B, NCTC, NCTC-PAR2, and HEK293 cells were transfected with increasing concentrations of the PAR4 N56A mECFP mutant or PAR4 mECFP. Whole cell lysates were prepared and resolved by Western blotting. C, fluorescent microscopy confirmed a loss of surface localization of PAR4 following mutation of N-glycosylation site (red arrows). Cells were visualized using a ×100 Plan Fluor objective. Scale bars = 10 μm. Blots and images are representative of three independent experiments.
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Related In: Results  -  Collection

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Figure 7: The presence of PAR2 allows N-linked glycosylation of PAR4, which promotes membrane localization. HEK293 cells were transfected with (A) PAR4 mECFP or PAR4 RAR mutant constructs. Prevention of de novo N-glycosylation was carried out by the addition of tunicamycin (2 μg/ml) to transfected cells as indicated for 16 h. B, NCTC, NCTC-PAR2, and HEK293 cells were transfected with increasing concentrations of the PAR4 N56A mECFP mutant or PAR4 mECFP. Whole cell lysates were prepared and resolved by Western blotting. C, fluorescent microscopy confirmed a loss of surface localization of PAR4 following mutation of N-glycosylation site (red arrows). Cells were visualized using a ×100 Plan Fluor objective. Scale bars = 10 μm. Blots and images are representative of three independent experiments.
Mentions: We then examined the role of PAR2 in regulating further post-translational modification of PAR4 in the context of receptor maturation and cell surface expression. N-Linked complex glycosylation is an important post-translational modification for efficient cell surface delivery of GPCRs (46). Analysis of the protein sequence of PAR4 identified an Asn-Xxx-serine/threonine N-glycosylation motif located at position 56 (N56DS) in the N terminus of the receptor (supplemental Fig. S1), suggesting that PAR4 may undergo N-linked glycosylation. To assess this, first, pharmacological inhibition of glycosylation was carried out using the GlcNAc phosphotransferase inhibitor tunicamycin, which prevents all N-linked glycosylation. Fig. 7A shows that pretreatment of HEK293 cells with tunicamycin eliminated the higher molecular mass form of PAR4, whereas the lower 65-kDa protein was still retained, with a minor intermediate band indicated. This effect was replicated in the R183AR to A183AA mutant, known to be preferentially expressed at the membrane. Experiments were then conducted using an N-terminal mutant of PAR4 (Asn56 → Ala56) to determine the effect of PAR2 upon protein species expression (Fig. 7B) and receptor localization (Fig. 7C). In control NCTC cells the PAR4 N56A mutant construct (Fig. 7B, top panel) was expressed as a single 65-kDa protein form that corresponded to wild type PAR4 mECFP. When expressed in NCTC-PAR2 cells (Fig. 7B, middle panel) a loss in the higher molecular mass protein form was observed in comparison to wild type PAR4 mECFP, however, the 65-kDa species was expressed alongside the slightly higher molecular mass form previously observed in the tunicamycin inhibition experiments. Similar results were obtained following expression in HEK293 cells (Fig. 7B, lower panel). Corresponding fluorescence microscopy images are shown in Fig. 7C. Lack of PAR4 cell surface expression was observed following expression of the PAR4 N56A mutant in NCTC-PAR2 cells.

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