Limits...
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.

Show MeSH

Related in: MedlinePlus

Retention of PAR4 in the ER. NCTC-2544 cells expressing pcDNA3.1 empty vector, PAR1 mEYFP, PAR2 mEYFP, or PAR4 mECFP (green) were treated with PKH26 red fluorescent cell linker dye to stain the plasma membrane (PM, red). Cells were fixed and treated with either (A) 4′,6-diamidino-2-phenylindole (DAPI) to identify nuclei (blue) or (B) ER marker (ER-Tracker Blue-White DPX Dyes, Molecular Probes) to identify the ER (blue). Cells were visualized using a ×100 Plan Fluor objective. Images were merged to highlight distinct plasma membrane, nucleus, or ER compartments. Scale bars = 10 μm. White arrows point to the plasma membrane, whereas red arrows are indicative of the intracellular/ER compartmentalization of PAR4. Image set representative of three separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3351358&req=5

Figure 1: Retention of PAR4 in the ER. NCTC-2544 cells expressing pcDNA3.1 empty vector, PAR1 mEYFP, PAR2 mEYFP, or PAR4 mECFP (green) were treated with PKH26 red fluorescent cell linker dye to stain the plasma membrane (PM, red). Cells were fixed and treated with either (A) 4′,6-diamidino-2-phenylindole (DAPI) to identify nuclei (blue) or (B) ER marker (ER-Tracker Blue-White DPX Dyes, Molecular Probes) to identify the ER (blue). Cells were visualized using a ×100 Plan Fluor objective. Images were merged to highlight distinct plasma membrane, nucleus, or ER compartments. Scale bars = 10 μm. White arrows point to the plasma membrane, whereas red arrows are indicative of the intracellular/ER compartmentalization of PAR4. Image set representative of three separate experiments.

Mentions: To monitor the expression level and localization of PAR4, the receptor was tagged at the C terminus with a monomeric variant form of enhanced cyan fluorescent protein (mECFP) and transiently expressed in keratinocyte-derived NCTC-2544 cells. These cells provided an ideal model for these investigations, due to the lack of endogenous PAR expression (44). The localization of PAR4 was initially monitored using fluorescence microscopy of NCTC-2544 cells transiently expressing PAR4 mECFP (Fig. 1A). In comparison to cells expressing either PAR1 mEYFP or PAR2 mEYFP, PAR4 mECFP was largely retained inside the cell with only weak membrane localization observed. Further microscopy in cells treated with an ER tracker dye (Fig. 1B) highlighted that PAR4 mECFP was predominantly retained in the ER.


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)

Retention of PAR4 in the ER. NCTC-2544 cells expressing pcDNA3.1 empty vector, PAR1 mEYFP, PAR2 mEYFP, or PAR4 mECFP (green) were treated with PKH26 red fluorescent cell linker dye to stain the plasma membrane (PM, red). Cells were fixed and treated with either (A) 4′,6-diamidino-2-phenylindole (DAPI) to identify nuclei (blue) or (B) ER marker (ER-Tracker Blue-White DPX Dyes, Molecular Probes) to identify the ER (blue). Cells were visualized using a ×100 Plan Fluor objective. Images were merged to highlight distinct plasma membrane, nucleus, or ER compartments. Scale bars = 10 μm. White arrows point to the plasma membrane, whereas red arrows are indicative of the intracellular/ER compartmentalization of PAR4. Image set representative of three separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Retention of PAR4 in the ER. NCTC-2544 cells expressing pcDNA3.1 empty vector, PAR1 mEYFP, PAR2 mEYFP, or PAR4 mECFP (green) were treated with PKH26 red fluorescent cell linker dye to stain the plasma membrane (PM, red). Cells were fixed and treated with either (A) 4′,6-diamidino-2-phenylindole (DAPI) to identify nuclei (blue) or (B) ER marker (ER-Tracker Blue-White DPX Dyes, Molecular Probes) to identify the ER (blue). Cells were visualized using a ×100 Plan Fluor objective. Images were merged to highlight distinct plasma membrane, nucleus, or ER compartments. Scale bars = 10 μm. White arrows point to the plasma membrane, whereas red arrows are indicative of the intracellular/ER compartmentalization of PAR4. Image set representative of three separate experiments.
Mentions: To monitor the expression level and localization of PAR4, the receptor was tagged at the C terminus with a monomeric variant form of enhanced cyan fluorescent protein (mECFP) and transiently expressed in keratinocyte-derived NCTC-2544 cells. These cells provided an ideal model for these investigations, due to the lack of endogenous PAR expression (44). The localization of PAR4 was initially monitored using fluorescence microscopy of NCTC-2544 cells transiently expressing PAR4 mECFP (Fig. 1A). In comparison to cells expressing either PAR1 mEYFP or PAR2 mEYFP, PAR4 mECFP was largely retained inside the cell with only weak membrane localization observed. Further microscopy in cells treated with an ER tracker dye (Fig. 1B) highlighted that PAR4 mECFP was predominantly retained in the ER.

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