The role of palmitoylation in signalling, cellular trafficking and plasma membrane localization of protease-activated receptor-2

Abstract

Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. This irreversible activation mechanism leads to rapid receptor desensitization by internalisation and degradation. We have explored the role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Experiments using the palmitoylation inhibitor 2-bromopalmitate indicated that palmitate addition is important in trafficking of PAR2 endogenously expressed by prostate cancer cell lines. This was supported by palmitate labelling using two approaches, which showed that PAR2 stably expressed by CHO-K1 cells is palmitoylated and that palmitoylation occurs on cysteine 361. Palmitoylation is required for optimal PAR2 signalling as Ca²⁺ flux assays indicated that in response to trypsin agonism, palmitoylation deficient PAR2 is ∼9 fold less potent than wildtype receptor with a reduction of about 33% in the maximum signal induced via the mutant receptor. Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. We also show that receptor palmitoylation occurs within the Golgi apparatus and is required for efficient agonist-induced rab11a-mediated trafficking of PAR2 to the cell surface. Palmitoylation is also required for receptor desensitization, as agonist-induced β-arrestin recruitment and receptor endocytosis and degradation were markedly reduced in CHO-PAR2-C361A cells compared with CHO-PAR2 cells. These data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor.

Figure 1. Inhibition of palmitoylation reduces cell surface expression of endogenous PAR2.

A, Upper panel; schematic representation of the structure of human PAR2 including the tethered ligand sequence (underline) within the amino terminal domain, C361 within a consensus palmitoylation motif present in carboxyl terminal domain, seven transmembrane (TM1 to TM7) domains, and a disulfide bond linking TM3 and the second extracellular loop (dotted line). Lower panel; protein sequence alignment of seventh transmembrane and carboxyl terminal residues of arginine vasopressin receptor-2 (V2R; NM_000054; residues 329–371), β2-adrenergic receptor (β2R; NM_000024.5; residues 329–413), PAR1 (NM_001992.3; residues 375–425), PAR2 (NM_005242.4; residues 347–397), PAR3 (NM_004101.2; residues 362–374) and PAR4 (NM_003950.2; residues 344–385). The known palmitoylation sites of V2R and β2R and the consensus palmitoylation sites of PAR1 and PAR2 are boxed. B, Western blot analysis of lysates from CHO cells stably transfected with vector or PAR2-myc probed with anti-PAR2 antibodies N19 and SAM11 and anti-myc and anti-GAPDH antibodies. *, non-specific band. C, Anti-PAR2 flow cytometry analysis of non-permeabilised CHO cells stably transfected with vector or PAR2-myc using antibodies N19 and SAM11. MFI values: CHO-PAR2 cells with N19 51.9±2.5; CHO-PAR2 cells with SAM11 41.6±3.2; signal from incubation of CHO-PAR2 cells with secondary antibody (2°), CHO-vector with antibody N19 (Vector N19) and CHO-vector with antibody SAM11 (Vector SAM11) were below 10⁰. D, Flow cytometry analysis of cell surface PAR2 endogenously expressed by non-permeabilised PC-3 cells using anti-PAR2 N19 and SAM11 antibodies. MFI values: N19 4.3±0.5; SAM11 2.3±0.9; secondary antibody (2°) 0.60±0.06. E, Graphical representation of the effect of blocking palmitoylation on cell surface expression of PAR2. Plasma membrane levels of PAR2, expressed endogenously by PC3, DU145 and 22Rv1 cells, were determined by flow cytometry using the anti-PAR2 N19 antibody. Non-permebilised cells were either treated with DMSO (negative control) or 2-bromopalmitate (+2BP) for 16 h. Secondary antibody only MFI values were subtracted from N19 values before calculation of the level of cell surface PAR2 present on 2BP treated cells relative to DMSO treated cells. Values were determined from 3 independent experiments and are shown as mean ± SD. **, P<0.001.

Figure 2. PAR2 is palmitoylated on cysteine 361.

A, Anti-myc and anti-GAPDH Western blot analysis of lysates from CHO cells stably expressing either vector, PAR2-myc or PAR2-C361A-myc. Before collection of lysates cells were either untreated or treated with the N-glycosylation inhibitor tunicamycin. *, non-specific band. B, Analysis of PAR2 palmitoylation using acyl-biotinyl exchange chemistry. Membrane fractions were collected from CHO-PAR2-myc and CHO-PAR2-C361A-myc cells either untreated or treated with 2-bromopalmitate (2BP) for 16 h. Free thiols on membrane proteins were blocked with NEM, endogenous palmitoyl groups were removed using hydroxylamine followed by sulphydryl-reactive biotinylation of liberated cysteines. Biotinylated proteins were isolated by streptavidin beads, eluted and subjected to anti-myc Western blot analysis to examine PAR2 palmitoylation. C, Analysis of PAR2 palmitoylation by examining cells metabolically labelled with an alkyne containing palmitate analogue (17-ODYA) followed by click chemistry. PAR2-myc and PAR2-C361A-myc CHO cells were incubated with 2-bromopalmitate for 16 h and cerulenin (5 µg/mL) for 1 h then labelled with 17-ODYA for 4 h, before collection of membrane preparations and reaction of biotin-azide to 17-ODYA labelled proteins via click chemistry. Biotinylated proteins were isolated using streptavidin beads and PAR2 palmitoylation was assessed by anti-myc Western blot analysis from bead elutes. D, Analysis of whether palmitoylation of PAR2 occurs exclusively on a cysteine residue and not via an amide linkage. Membrane preparations were collected from CHO-PAR2-myc and CHO-PAR2-C361A-myc 17-ODYA labelled cells. Labelled proteins were reacted with biotin-azide via click chemistry before incubation with 0.7 M hydroxylamine (H) to cleave thioester linkages or 50 mM tris pH 7.4 (T; control). Biotinylated proteins were isolated using streptavidin beads and PAR2 palmitoylation was assessed by anti-myc Western blot analysis from bead elutes. E, Comparison of intracellular Ca²⁺ mobilisation mediated by trypsin activation of PAR2-myc (○, EC50 5.3±0.5 nM) and PAR2-C361A-myc (□, EC50 44.3±0.8 nM) in stably expressing CHO cells. Experiments were performed in triplicate on 3 separate occasions and values are Mean +/− SD.

Figure 3. Palmitoylation of PAR2 at C361 is required for efficient cell surface receptor localization.

A, Images of CHO cells transiently co-transfected for 12 h with PAR2-mCherry (red) and PAR2-GFP (green). The merge image shows colocalization (yellow) of these two proteins. B, Images of CHO cells transiently co-transfected for 12 h with PAR2-mCherry and PAR2-C361A-GFP. The merge image shows colocalization (yellow) of these two proteins and regions where only PAR2-mCherry is expressed (red). Cells were analysed using a Zeiss LSM510 confocal microscope (63× oil immersion objective lens) and images were processed using MetaMorph software and displayed using CorelDraw X5. Images of cells are representative of three independent experiments. Scale bar, 10 µM.

Figure 4. Quantitative analysis of the effect of palmitoylation on cell surface expression of PAR2.

A, Anti-PAR2 N19 antibody flow cytometry analysis of non-permeabilised CHO-PAR2-myc and CHO-PAR2-C361A-myc cells treated with either DMSO (negative control) or 2BP for 16 h. Secondary antibody only MFI values were subtracted from N19 values and these are shown relative to values from CHO-vector untreated cells. Values were determined from 3 independent experiments and are shown as Mean ± SD. B, (Left panel) Examination of plasma membranePAR2 levels by cell surface biotinylation. Live cells were reacted with membrane impermeant EZ-link NHS-SS-biotin. Plasma membrane (PM) and intracellular (IC) fractions were collected from whole cell lysates and subjected to anti-myc, anti-GAPDH (control for IC fraction) and anti-β1 integrin (control for PM fraction) Western blot analysis. The data are representative of 3 independent experiments. (Right panel) Graphical representation of densitometric analysis of data from these 3 experiments. Values of PM PAR2 were normalised to β1 integrin signal and are displayed as Mean ± SD. C, Anti-PAR2 N19 antibody flow cytometry analysis of DU145 cells transiently transfected with vector, PAR2-GFP or PAR2-C361A-GFP. MFI values from 3 independent experiments were used to calculate fold change of cell surface PAR2 and PAR2-C361A relative to vector transfected cells and are displayed as Mean ± SD. **, P<0.05; ***, P<0.001.

Figure 5. PAR2 agonism stimulates palmitate incorporation which occurs during secretory trafficking in pre-medial Golgi vesicles.

A, CHO-PAR2-myc cells preincubated with cerulenin for 1 h were labelled with 17-ODYA for 1.5 h in the presence or absence of PAR2-AP (100 µM) for the indicated times. Membrane preparations were collected and biotin-azide reacted to 17-ODYA labelled proteins via click chemistry. Biotinylated proteins were isolated using streptavidin beads. PAR2 palmitoylation was assessed by anti-myc Western blot analysis of bead elutes. B, CHO-PAR2-myc cells preincubated with cerulenin were labelled with 17-ODYA for 2 h in the presence or absence of PAR2 AP (100 µM). Nocodazole (20 µg/mL; NOC) was added to medium 15 min prior to labelling with 17-ODYA and monensin (10 µM; MON) and brefeldin A (5 µg/mL; BFA) were added to medium 30 min prior to labelling with 17-ODYA. Labelled proteins were reacted to biotin-azide via click chemistry and biotinylated proteins isolated using streptavidin beads. Palmitoylated PAR2 was detected by anti-Myc Western blot analysis of bead elutes. Data shown in B is from cropped lanes from a single Western blot analysis. The data are representative of three independent experiments.

Figure 6. Palmitoylation of PAR2 is required for efficient rab11a-mediated repopulation of the cell surface in response to agonist stimulation.

CHO-PAR2-myc and CHO-PAR2-C361A-myc cells transiently transfected with vector, rab11a-GFP or dominant negative rab11a-S25N-GFP were treated with 50 nM trypsin for 15 min to remove cell surface PAR2. Cells were then washed and incubated with trypsin-free DMEM for the indicated times to allow repopulation of PAR2 at the cell surface. Plasma membrane PAR2 was measured by flow cytometry using the anti-PAR2 N19 antibody. MFI values were used to calculate percentage of cell surface PAR2 relative to vector cells. Experiments were performed in triplicate on 3 independent occassions. Data are displayed as mean ± SD. Statistical significant differences - 30 min: PAR2+vector 36.8±1.6%, PAR2+rab11a-GFP 65.5%, P<0.0001; PAR2-C361A+vector 22.7±3.5%, PAR2-C361A+rab11a-GFP 33.4±4.0%, P<0.05; PAR2-C361A+rab11a-GFP 33.4±4.0%, PAR2+rab11a-GFP 65.5%, P<0.0001, compared with PAR2-C361A+rab11a-GFP.

Figure 7. Mutation of PAR2 C361 alters agonist-induced recruitment of β-arrestin-1 delays β-arrestin-2.

Confocal microscopy analysis of untreated live cells and at 5 and 15 min after agonism with PAR2 AP (100 µM). A, CHO cells transiently co-expressing PAR2-mCherry (red) and β-arrestin-1-GFP (green) or β-arrestin-2-GFP (green). The merge image highlights colocalization (yellow) of PAR2 and β-arrestins. B, CHO cells transiently co-expressing PAR2-C361A-mCherry (red) and β-arrestin-1-GFP (green) or β-arrestin-2-GFP (green). The merge image highlights colocalization (yellow) of PAR2 and β-arrestins. Cells were analysed using a Zeiss LSM510 confocal microscope (63× oil immersion objective lens) and images were processed using MetaMorph software and displayed using CorelDraw X5. Images of cells are representative of three independent experiments. Scale bar, 10 µM.

Figure 8. PAR2 C361 is required for efficient agonist-induced receptor endocytosis and degradation.

A, CHO-PAR2-myc and CHO-PAR2-C361A-myc cells labelled with membrane impermeant EZ-link NHS-SS-biotin were treated with PAR2 AP (100 µM) for the indicated times to induce receptor internalisation. Protein degradation was blocked by incubation of cells with the proteasome inhibitor MG132. Residual cell surface biotin was removed by washing with MeSNA and internalised biotin-labelled protein was isolated from whole cell lysates using streptavidin beads. Anti-myc Western blot analysis was performed on bead eluates and input lysates. Data are representative of 3 independent experiments with graphical representation of densitometry analysis of these data shown in the right panel. Percentage of internalised receptor was calculated by dividing the value for internalised PAR2 at each time point by the value for total surface PAR2 which was determined from an experiment performed in parallel in which cells were not treated with AP or MeSNA. Values are displayed as Mean ± SD. B, CHO-PAR2-myc and CHO-PAR2-C361A-myc cells were treated with PAR2 AP (100 µM) for the indicated times in the presence of 70 µM cycloheximide, to prevent de novo protein synthesis. Lysates were examined by anti-myc and anti-GAPDH Western blot analysis to assess the levels of PAR2 remaining after receptor agonism. Data are representative of 3 independent experiments with graphical representation of densitometry analysis of these data shown in the right panel. Values are displayed as Mean ± SD. PAR2 endocytosis and degradation were significant compared with PAR2-C361A; P<0.001.

Figure 9. PAR2 palmitoylation is required for optimal receptor signalling, cellular trafficking and plasma membrane expression.

Unpalmitoylated PAR2 located in Golgi vesicles (1). Palmitoylation occurs within the cis- to medial-Golgi (2). This anchors the carboxyl domain of PAR2 to the inner leaflet of cell membranes likely resulting in the formation of an intracellular eighth α-helix (H8) required for effective signalling (2). Interactions of palmitoylated PAR2 with the GTPase rab11a promotes agonist induced receptor repopulation of the cell surface (3). Activation of PAR2 (4) enhances palmitoylation of Golgi localised PAR2 (5) and, reciprocally, receptor palmitoylation is required for efficient PAR2 signal transduction (6). Receptor agonism induces β-arrestin translocation to the plasma membrane (7) which mediates non-G protein signal transduction and receptor internalisation (8). Internalised PAR2 is trafficked and sorted for lysosomal degradation via early and late endosomes (9).