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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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Enhanced PAR4-mediated [3H]inositol phosphate accumulation in the presence of PAR2. NCTC-2544 and NCTC-PAR2 cells transiently expressing PAR4 mECFP were serum starved with serum-free growth medium supplemented with 0.25 μCi of myo-[2-3H]inositol for 24 h. Cells were pretreated with 10 mm lithium chloride for 15 min prior to stimulation with AYPGKF-NH2 as indicated for 45 min. Total inositol phosphate (InsP1–4) accumulation was measured via anion exchange. The data are representative values performed in triplicate (mean ± S.E.) over three independent experiments (**, p = <0.01).
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Figure 8: Enhanced PAR4-mediated [3H]inositol phosphate accumulation in the presence of PAR2. NCTC-2544 and NCTC-PAR2 cells transiently expressing PAR4 mECFP were serum starved with serum-free growth medium supplemented with 0.25 μCi of myo-[2-3H]inositol for 24 h. Cells were pretreated with 10 mm lithium chloride for 15 min prior to stimulation with AYPGKF-NH2 as indicated for 45 min. Total inositol phosphate (InsP1–4) accumulation was measured via anion exchange. The data are representative values performed in triplicate (mean ± S.E.) over three independent experiments (**, p = <0.01).

Mentions: Finally, the effect of enhanced cell surface expression of PAR4 in the presence of PAR2 was explored further in relationship to PAR4-mediated cell signal transduction. As shown in Fig. 8, NCTC-2544 and NCTC-PAR2 cells transiently expressing PAR4 mECFP produced an increase in basal inositol phosphate generation (NCTC, 4.59 ± 0.45; NCTC-PAR2, 8.28 ± 0.65-fold of basal) compared with mock transfected cells. When each of these cell systems was treated with the PAR4-specific activating peptide, AYPGKF-NH2, a further increase in the inositol phosphate response was observed. When PAR4 was expressed in NCTC-PAR2 cells total inositol phosphate (InsP1–4) accumulation (50 μm agonist; 22.47 ± 0.45 and 100 μm agonist; 21.16 ± 2.62-fold of basal) was substantially greater than observed following expression of PAR4 in NCTC-2544 cells (50 μm agonist; 7.80 ± 0.46 and 100 μm agonist; 9.47 ± 2.45-fold of basal).


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)

Enhanced PAR4-mediated [3H]inositol phosphate accumulation in the presence of PAR2. NCTC-2544 and NCTC-PAR2 cells transiently expressing PAR4 mECFP were serum starved with serum-free growth medium supplemented with 0.25 μCi of myo-[2-3H]inositol for 24 h. Cells were pretreated with 10 mm lithium chloride for 15 min prior to stimulation with AYPGKF-NH2 as indicated for 45 min. Total inositol phosphate (InsP1–4) accumulation was measured via anion exchange. The data are representative values performed in triplicate (mean ± S.E.) over three independent experiments (**, p = <0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351358&req=5

Figure 8: Enhanced PAR4-mediated [3H]inositol phosphate accumulation in the presence of PAR2. NCTC-2544 and NCTC-PAR2 cells transiently expressing PAR4 mECFP were serum starved with serum-free growth medium supplemented with 0.25 μCi of myo-[2-3H]inositol for 24 h. Cells were pretreated with 10 mm lithium chloride for 15 min prior to stimulation with AYPGKF-NH2 as indicated for 45 min. Total inositol phosphate (InsP1–4) accumulation was measured via anion exchange. The data are representative values performed in triplicate (mean ± S.E.) over three independent experiments (**, p = <0.01).
Mentions: Finally, the effect of enhanced cell surface expression of PAR4 in the presence of PAR2 was explored further in relationship to PAR4-mediated cell signal transduction. As shown in Fig. 8, NCTC-2544 and NCTC-PAR2 cells transiently expressing PAR4 mECFP produced an increase in basal inositol phosphate generation (NCTC, 4.59 ± 0.45; NCTC-PAR2, 8.28 ± 0.65-fold of basal) compared with mock transfected cells. When each of these cell systems was treated with the PAR4-specific activating peptide, AYPGKF-NH2, a further increase in the inositol phosphate response was observed. When PAR4 was expressed in NCTC-PAR2 cells total inositol phosphate (InsP1–4) accumulation (50 μm agonist; 22.47 ± 0.45 and 100 μm agonist; 21.16 ± 2.62-fold of basal) was substantially greater than observed following expression of PAR4 in NCTC-2544 cells (50 μm agonist; 7.80 ± 0.46 and 100 μm agonist; 9.47 ± 2.45-fold of basal).

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