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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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Membrane localization of PAR4 mECFP in HEK293 cells. PAR4 mECFP was transiently transfected into HEK293 cells that endogenously express PAR2. A, localization of PAR4 at the cells surface is shown by co-localization with a plasma membrane marker (white arrows). Cells were visualized using a ×100 Plan Fluor objective. Scale bars = 10 μm. B, the protein band pattern of PAR4 expression was further assessed by Western blot of transfected whole cell lysates followed by subcellular fractionation (C) as previously described. Images and blots are representative of three separate experiments.
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Figure 5: Membrane localization of PAR4 mECFP in HEK293 cells. PAR4 mECFP was transiently transfected into HEK293 cells that endogenously express PAR2. A, localization of PAR4 at the cells surface is shown by co-localization with a plasma membrane marker (white arrows). Cells were visualized using a ×100 Plan Fluor objective. Scale bars = 10 μm. B, the protein band pattern of PAR4 expression was further assessed by Western blot of transfected whole cell lysates followed by subcellular fractionation (C) as previously described. Images and blots are representative of three separate experiments.

Mentions: The localization of PAR4 was further explored in HEK293 cells, which have an endogenous level of PAR2 (Fig. 5). When expressed, PAR4 mECFP was observed both at the plasma membrane and in intracellular compartments (Fig. 5A), which when resolved by Western blot (Fig. 5B) identified similar protein species as in NCTC-2544 cells, representative receptor populations were expressed at the cell surface and in ER/endosomal compartments (Fig. 5C).


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)

Membrane localization of PAR4 mECFP in HEK293 cells. PAR4 mECFP was transiently transfected into HEK293 cells that endogenously express PAR2. A, localization of PAR4 at the cells surface is shown by co-localization with a plasma membrane marker (white arrows). Cells were visualized using a ×100 Plan Fluor objective. Scale bars = 10 μm. B, the protein band pattern of PAR4 expression was further assessed by Western blot of transfected whole cell lysates followed by subcellular fractionation (C) as previously described. Images and blots are representative of three separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Membrane localization of PAR4 mECFP in HEK293 cells. PAR4 mECFP was transiently transfected into HEK293 cells that endogenously express PAR2. A, localization of PAR4 at the cells surface is shown by co-localization with a plasma membrane marker (white arrows). Cells were visualized using a ×100 Plan Fluor objective. Scale bars = 10 μm. B, the protein band pattern of PAR4 expression was further assessed by Western blot of transfected whole cell lysates followed by subcellular fractionation (C) as previously described. Images and blots are representative of three separate experiments.
Mentions: The localization of PAR4 was further explored in HEK293 cells, which have an endogenous level of PAR2 (Fig. 5). When expressed, PAR4 mECFP was observed both at the plasma membrane and in intracellular compartments (Fig. 5A), which when resolved by Western blot (Fig. 5B) identified similar protein species as in NCTC-2544 cells, representative receptor populations were expressed at the cell surface and in ER/endosomal compartments (Fig. 5C).

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