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Am J Physiol Cell Physiol 293: C346-C357, 2007. First published April 18, 2007; doi:10.1152/ajpcell.00010.2007
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RECEPTORS AND SIGNAL TRANSDUCTION

Differential effects of beta-arrestins on the internalization, desensitization and ERK1/2 activation downstream of protease activated receptor-2

P. Kumar,1 C. S. Lau,2 M. Mathur,3 P. Wang,1 and K. A. DeFea1,2,3

1Division of Biomedical Sciences, 2Biochemistry and Molecular Biology Program, 3Cell, Molecular and Developmental Biology Program, University of California, Riverside, Riverside, California

Submitted 6 January 2007 ; accepted in final form 14 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
beta-Arrestins-1 and 2 are known to play important roles in desensitization of membrane receptors and facilitation of signal transduction pathways. It has been previously shown that beta-arrestins are required for signal termination, internalization, and ERK1/2 activation downstream of protease-activated-receptor-2 (PAR-2), but it is unclear whether they are functionally redundant or mediate specific events. Here, we demonstrate that in mouse embryonic fibroblasts (MEFs) from beta-arrestin-1/2 knockout mice, G{alpha}q signaling by PAR-2, as measured by mobilization of intracellular Ca2+, is prolonged. Only expression of beta-arrestin-1 shortened the signal duration, whereas either beta-arrestin-1 or 2 was able to restore PKC-induced receptor desensitization. beta-arrestin-1 also mediated early, while beta-arrestin-2 mediated delayed, receptor internalization and membrane-associated ERK1/2 activation. While beta-arrestin-1 colocalized with a lysosomal marker (LAMP-1), beta-arrestin-2 did not, suggesting a specific role for beta-arrestin-1 in lysosomal receptor degradation. Together, these data suggest distinct temporal and functional roles for beta-arrestins in PAR-2 signaling, desensitization, and internalization.

arrestins; PAR-2; protease-activated-receptor; G protein-coupled receptor; ERK1/2

beta-ARRESTINS 1 AND 2 ARE MULTIFACETED proteins that can promote signal termination of many heterotrimeric G protein-initiated signals, as well as regulate the localization and activity of downstream signaling molecules and facilitate chemotaxis (4, 11, 12, 16–18, 21, 23, 25, 26, 39, 43). Signal termination can be broken down into two parts: desensitization of the receptor and removal of receptor from the cell surface by endocytosis. G protein-coupled receptor (GPCR) desensitization is usually defined as a molecular event that renders the receptor insensitive to further activation and/or attenuates the initial signal generated. beta-arrestins participate in both processes, by binding to receptors (usually via phospho-ser/thr residues in the C terminus and third cytosolic loop) and uncoupling them from their cognate G-proteins (28) and by binding to both clathrin and the clathrin adaptor proteins, AP-1 and AP-2, to facilitate endocytosis (24, 32).

Protease-activated-receptor-2 (PAR-2) is a GPCR that mediates a diverse array of cellular processes in response to serine proteases. Previous studies on PAR-2 have demonstrated that it undergoes agonist-induced (homologous) desensitization and internalizes via a beta-arrestin-dependent mechanism. Both processes are impaired in mouse embryonic fibroblasts (MEFs) from beta-arrestin double knockout mice; internalization is also blocked by a dominant-negative mutant of beta-arrestin-1 (15, 38). Furthermore, overexpression of either beta-arrestin-1 or -2 is reported to increase accumulation of IP3 (a G{alpha}q signaling intermediate), suggesting that beta-arrestins terminate IP3 generation through uncoupling of the receptor from G{alpha}q. For desensitization and internalization of some GPCRs, the two beta-arrestins are interchangeable, whereas for others they have distinct roles (1, 2, 4, 37, 44). Previous studies have also demonstrated that PAR-2 requires both beta-arrestins for efficient ERK1/2 activation and chemotaxis (13, 17, 18, 38), suggesting they may regulate different events in PAR-2 signaling and signal termination. Furthermore, several lines of evidence suggest PKC activation can promote desensitization and internalization of PAR-2, leading to the hypothesis that either or both beta-arrestins might bind to PKC phosphorylated sites in the PAR-2 C terminus (7, 13). Upon agonist-induced endocytosis, many receptors are recycled back into the plasma membrane; however, since the physiological agonists of PAR-2 are serine proteases that remove the extreme N terminus, rendering it unusable, most is degraded rather than recycled. Restoration of surface PAR-2 levels requires exocytosis from Golgi stores, by a process involving the small GTPase-Rab11 (7, 34). Whether beta-arrestins play a role in receptor exocytosis has not been examined.

Because previous studies demonstrating a requirement for beta-arrestins in PAR-2 internalization were done in MEFs from mice lacking both beta-arrestins or in cells stably transfected with a dominant negative mutant of beta-arrestin-1 that can block both beta-arrestin-1 and -2 (15, 38), it is unclear whether the two beta-arrestins regulate distinct processes during PAR2 desensitization, internalization, and signaling. In one study, overexpression of either beta-arrestin-1 or -2 attenuated accumulation of the second messenger IP3, suggesting that either one can promote receptor uncoupling from G{alpha}q (38), while in another study, knockdown of either beta-arrestin-1 or -2 nearly abolished ERK1/2 activation, suggesting that both are required for signaling (18). Furthermore, no studies have investigated their role in heterologous desensitization such as that promoted by PKC activation.

Because beta-arrestins were first identified as mediators of signal termination and receptor endocytosis, it has often been assumed that their role in other downstream signaling events such as ERK1/2 activation is secondary to their ability to mediate endocytosis. However, recent studies have revealed that beta-arrestins can elicit signals, independent of G protein coupling, suggesting that their roles as mediators of signal termination can be separated from their roles as facilitators of signal transduction. In this paper, we probe the role of individual beta-arrestins in PAR-2 desensitization, internalization, and ERK1/2 activation. We show that not only can the individual arrestins promote their "arresting" (desensitizing) effects differently but can also mediate internalization and downstream signaling of a receptor in different ways.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Rabbit anti-PAR2 B5 antibody was a generous gift from Dr. Morley Hollenberg (University of Calgary, Calgary, Canada). Rabbit anti-LAMP1, rabbit anti-ERK1, and mouse PAR-2 antibodies were obtained from Santa Cruz Biotechnology, mouse anti-EEA1 was obtained from BD Transduction Laboratories, anti-phospho-ERK1/2 was obtained from Cell Signaling Technologies, and anti-FLAG M2 antibody was obtained from Sigma. Fluorescent secondary antibodies anti-rabbit Alexa 488/546/633 and anti-mouse Alexa 488/546 were obtained from Molecular probes, 2-furoyl-LIGRL (2-fAP) was synthesized by Genemed, trypsin was obtained from Worthington Chemicals, Fura2-AM and Pluronic F127 were obtained from Molecular Probes, PMA was obtained from Sigma-Aldrich, FLAG-tagged beta-arrestin 1 and 2 constructs, and anti-beta-arrestin-1/2 antibodies were a kind gift from Dr. Robert Lefkowitz (Duke University Medical Center), Cell Stripper & 1x PBS were from GIBCO BRL.

Cell culture. Mouse embryonic fibroblasts wild type (MEF WT), beta-arrestin double knockout (MEF betaarrDKO), beta-arrestin-1 (MEF DKO + betaArr1), beta-arrestin-2 (MEF DKO + betaArr2) were a gift from Dr. Robert Lefkowitz, and human breast cancer cells (MDA-MB 468) and Chinese hamster ovary (CHO) cells were obtained from ATCC. MEFs and MDA MB-468 cells were grown in DMEM medium supplemented with 10% cosmic calf serum and 10 U/ml penicillin/streptomycin, and CHOs were grown in DMEM/F-12 supplemented with 10% FCS and Pen/Strep. All cells were maintained at 37°C with 5% CO2. For passaging, cells were detached using cell stripper solution. All transfections were done using lipofectamine and Plus Reagent (Invitrogen) using standard procedures. For CHO cells stably transfected with PAR-2 GFP, cells were selected in G418 for 2 wk until green fluorescent protein (GFP)-positive colonies formed. To avoid clonal cell artifacts, all positive colonies were pooled and selected for PAR-2-GFP expression using a FACS-Aria. Stable cell lines were maintained in the absence of G418 for at least before all experiments.

Flow cytometry analysis of surface expression of PAR2. Flow cytometry was used to quantify removal of PAR2 from the plasma membrane and to thereby determine the rate of endocytosis. Cells were treated with 100 nM 2-fAP for 0–60 min, washed in ice-cold PBS, detached with ice cold cell stripper, and incubated with a 1:500 dilution of anti-PAR-2 (B5 antibody) in DMEM + 1% BSA for 2 h on ice followed by FITC-conjugated secondary antibody for 1 h on ice in dark. Surface cell fluorescence was determined by flow cytometry using a BD FACScan flow cytometer. Cellquest Pro software was used to determine the mean fluorescence and number of FITC-positive cells.

Immunofluorescence and confocal microscopy. Cells were seeded onto collagen-coated coverslips (3 x 104 cells/coverslip) and allowed to grow overnight, after which they were transfected using lipofectamine and Plus Reagent with the appropriate plasmids, according to manufacturer's instructions. Twenty-four hours posttransfection, media was changed to serum-free DMEM, and cells were treated with 100 nM 2-fAP or 50 nM trypsin for 0–60 min, after which they were washed in ice-cold 1x PBS, fixed in 4% neutral buffered formalin and immunostained, as described previously (17). Antibody concentrations were as follows: anti-Flag (1:1,000), anti-LAMP-1 (1:500), anti-EEA-1 (1:500), Texas Red-Transferrin (10 µg/ml), anti-PAR-2 (1:250). They were then mounted using DAKO Cytomation fluorescent mounting medium onto slides and imaged using a Zeiss LSM 510 confocal microscope. All images were collected and analyzed using LSM Browser software.

Single-cell calcium concentration measurements. PAR2-GFP-transfected MEFs or untransfected MDA MB-468 cells were incubated in physiological salt solution (PSS; 137 mM NaCl, 4.7 mM KCl, 0.56 mM MgCl2, 2 mM CaCl2, 1 mM Na2HPO4, 10 mM HEPES, 2 mM L-glutamine and 5. 5 mM D-glucose pH 7.4), containing 0.1% BSA and a 1:1 mixture of 5 µM Fura-2/AM and 50% Pluronic F-127 detergent for 40 min at 37°C. They were then washed and mounted in a perfusion chamber containing 1.5 ml of PSS-BSA at 37°C on the stage of a Nikon TE300 microscope. Agonists were directly added to the bath. Cells were observed with a x40 objective, and fluorescence was detected in individual cells using a Nikon video camera and a video microscopy acquisition program (Metafluor). Fluorescence was measured at 340 and 380 nm excitation and 510 nm emission. The ratio of the fluorescence at the two excitation wavelengths, which is proportional to the [Ca+2], was determined. For dose-response experiments, cells were exposed to increasing concentrations of agonist; for all other experiments, a single concentration of agonist was used [50 nM trypsin and 100 nM 2furoyl-Lys-Iso-Gly-Arg-Leu (2fAP)]. To examine desensitization, cells were pretreated with 1 µM PMA for 20 min before agonist challenge. All readings were followed by an application of the calcium ionophore, ionomycin, to determine maximum Ca2+ levels. The concentration of intracellular Ca2+ in response to the agonist was determined using the Grynkiewitz equation (20).

Protein analysis and Western blot analysis. For all protein analysis, Laemmli sample buffer was added to each sample (Final concentration: 50 mM Tris-HCl pH 6.8, 2.5% SDS, 10% glycerol, 5% beta-mercaptoethanol, 0.1% bromophenol blue). Equal amounts of protein were loaded onto polyacrylamide minigels (10% for beta-arrestin analysis, 12% for ERK analysis) and run for ~2 h at 100 V. Proteins were transferred to polyvinylidene difluoride membrane for 30 min, blocked in TBS+1% fish gelatin, and incubated with primary antibody solutions [pERK: 1:1,000 + tERK 1:1,000 and beta-arrestin-1+2 (A1CT, 1:500)] overnight at 4°C. Blots were washed 3 times in Tris-buffered saline supplemented with 1% Tween-20, followed by incubation with either Alexa680- or IR800-conjugated secondary antibodies (1:45,000) for 1 h at room temperature. Blots were washed 3 times in TBS-Tween and analyzed using the LICOR-Odyssey infrared imaging system. Odyssey software was used to calculate integrated intensities of each band for quantification. Western blot analysis shown are representative of at least 3 experiments.

pERK1/2 assays. For total phospho-ERK analysis, cells (~80% confluent) were serum starved in DMEM + 0.1% BSA overnight and treated with 50 nM trypsin for 0–60 min, then lysed in RIPA buffer (1x PBS containing 1% Triton-X 100, 2 mM EDTA, 10 mM NaF, 5 mM activated Na3VO4, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 10 µg/ml benzamidine and 2 mM PMSF). Ten micrograms of protein from each time point were analyzed by SDS-PAGE followed by Western blot analysis with phospho-ERK1/2 antibody and a total ERK1/2 antibody, as described above. For determination of membrane vs. nuclear ERK1/2, cells were treated for 0–90 min and ruptured by homogenization with a glass dounce in hypotonic lysis buffer (10 mM triethanolamine-acetic acid, pH 7.6, 250 mM sucrose, 1 mM EGTA, 10 mM NaF, 2 mM Na3VO4, 1 mM PMSF, 1 mM DTT, plus 10 µg/ml of leupeptin, aprotinin, and benzamidine). Nuclear fractions were collected by centrifugation at 500 g, and pellets were resuspended in Laemmli sample buffer. Membrane fractions were collected by centrifugation of low-speed supernatants at 100,000 g for 1 h. Pellets were resuspended in Laemmli sample buffer. SDS-PAGE and Western blot analysis was conducted as described above.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Role of beta-arrestins in receptor-mediated desensitization. Upon proteolytic activation by trypsin, PAR2 can couple to G{alpha}q, which activates phospholipase Cbeta (PLCbeta), leading to PIP2 hydrolysis and the generation of IP3 and DAG. IP3 mobilizes Ca2+ from intracellular stores; for this reason, Ca2+ mobilization is frequently used as a direct readout of PAR-2/G{alpha}q-protein coupling and PIP2 hydrolysis (7, 8, 14, 30, 31). Peptides corresponding to the tethered ligand (SLIGRL or SLIGKV) can be used to activate PAR-2 in the absence of proteolytic cleavage but exhibit very low affinity and are typically used in the high micromolar range (3, 8). More recently, higher-affinity, synthetically modified peptides, such as 2fAP have been developed (27), which is used in this study for nonproteolytic PAR-2 activation. Early studies on PAR-2 signaling demonstrated that treatment with agonist peptides renders the receptor insensitive to a second agonist-induced G{alpha}q coupling, as assayed by mobilization of intracellular Ca2+ (8). Heterologous activation of PKC by phorbol esters was also shown to attenuate PAR-2-induced Ca2+ mobilization, and mutation of putative PKC phosphorylation sites abolishes PMA-induced desensitization (7, 13). To examine whether there was a differential requirement for either beta-arrestin-1 or 2 in PAR-2 desensitization, we measured trypsin-induced Ca2+ mobilization in mouse embryonic fibroblasts from wt (MEFwt) or beta-arrestin-1/2–/– mice (MEF DKO), and MEF DKO expressing endogenous levels of either beta-arrestin1 (DKO+betaarr1) or beta-arrestin2 (DKO+betaarr2). These cells have been characterized previously and used by numerous laboratories to assess beta-arrestin dependence in different cellular events (22, 38, 42). Expression of PAR-2 is relatively low in these cells (38); thus, we expressed PAR-2 GFP, which has previously been demonstrated to traffic and signal like the endogenous receptor (15), to monitor Ca2+ mobilization.

We first determined dose-dependent Ca2+ mobilization in response to proteolytic (trypsin) and nonproteolytic (2fAP) PAR-2 agonists in MEFwt and MEF betaarrDKO, transfected with PAR-2GFP to establish whether initial PAR-2 responses were altered by the absence of beta-arrestins. Additionally, dose responses of transfected MEFs were compared with that observed in a breast cancer cell line, MDA MB-468, which expresses high levels of endogenous PAR-2 and has been used to characterize PAR-2 signaling in our laboratory (17, 18, 43). Consistent with reports by others, transfected PAR-2GFP and endogenous PAR-2 promoted similar dose-dependent increases in intracellular Ca2+ levels in response to trypsin and 2fAP (Fig. 1). Furthermore, both agonists induced a similar dose-dependent increase in Ca2+ levels in the presence and absence of beta-arrestins (compare MEFwt to MEFbetaarrDKO, Fig. 1), suggesting that beta-arrestins are not necessary for PAR-2/ G{alpha}q coupling. As reported by others, nonproteolytic agonists have a lower efficiency but are equally efficacious at promoting Ca2+ mobilization (8). It is noteworthy that the response to 2fAP was slightly left-shifted in MEFbetaarrDKO compared with MEFwt, which may reflect the fact that in the absence of beta-arrestins, the soluble peptide can continue to activate the receptor. This same left shift would not be expected for trypsin because the receptor cannot be reused once it has been proteolytically cleaved. For all subsequent studies, 50 nM trypsin and 100 nM 2fAP were used; at these concentrations, the responses in both transfected MEF cell lines and MDA MB-468 cells were comparable (Fig. 1).


Figure 1
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  		Fig. 1. Dose response of Ca2+ mobilization to trypsin and 2furoyl-Lys-Iso-Gly-Arg-Leu (2fAP). Mobilization in mouse embryonic fibroblasts from wt (MEFwt), beta-arrestin double knockout (MEFbetaarrDKO) [both transfected with protease-activated-receptor-2 (PAR-2GFP)] and MDA MB-468 cells were loaded with Fura 2AM, treated with increasing concentrations of trypsin (A) or 2Fap (B), and the ratio of fluorescence at 340/380 nM was determined. Maximum concentrations of intracellular Ca2+ for each concentration were calculated according to the Grynkiewitz equation and plotted as a function of Log [agonist].

 
We then compared the duration of Ca2+ mobilization in response to PAR-2, and the effect of PMA on PAR-2 stimulated intracellular Ca2+ levels in MEFwt (Fig. 2, A and B), MEFbetaarrDKO (Fig. 2, C and D), DKO+betaarr1 (Fig. 2, E and F) and DKO+betaarr2 (Fig. 2, G and H). While the absence of either or both beta-arrestins did not significantly affect the maximum intracellular Ca2+ concentration, the duration of the Ca2+ response was increased in MEF DKO, suggesting defective G-protein uncoupling (Fig. 2J). Expression of beta-arrestin-1 (MEFDKO+betaarr1) decreased the Ca2+ signal duration to that of MEFwt, while expression of beta-arrestin-2 (MEFDKO+betaarr2) did not, implying that initial PAR-2/G protein-uncoupling is primarily mediated by beta-arrestin-1 (Fig. 2J). Next, we examined heterologous desensitization by pretreating cells with PMA for 20 min before the addition of PAR2 agonist. As was previously reported, Ca2+ mobilization in wtMEF was inhibited by greater than 70% by PMA treatment, while Ca2+ mobilization by ionomycin was unaffected (Fig. 2, B and K). MEFbetaarrDKO cells were resistant to PMA-induced receptor desensitization, while MEFDKO+betaarr1 (Fig. 2, F and K) and MEFDKO+betaArr2 (Fig. 2, H and K) were not, suggesting that both beta-arrestins are involved in mediating PMA-induced receptor desensitization.


Figure 2
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  		Fig. 2. beta-arrestins mediate PKC-induced PAR-2 desensitization and determine the duration of Ca2+ signaling. A–H: representative Ca2+ mobilization traces from Fura2/AM-loaded MEFwt (A and B), MEFbetaarrDKO (C and D), DKO+betaarr1 (E and F), and DKO+betaarr2 (G and H). Cells were pretreated with either vehicle (A, C, E, G) or 1 µM PMA (B, D, F, H) before addition of 50 nM trypsin, followed by the addition of a Ca2+ ionophore (ionomycin). I: Western blot analysis showing beta-arrestin levels in MEF wt, MEFbetaarrDKO, DKO+betaarr1, and DKO+betaarr2. J: average duration of the PAR-2-evoked Ca2+ signal in each MEF cell line was determined from 4 independent experiments and presented as means ± SE, P < 0.01. K: average maximal PAR-2-evoked Ca2+ responses in each MEF cell line, with and without PMA pretreatment, were calculated from 3 independent experiments and presented as means ± SE, P < 0.05. Statistical significance was determined by ANOVA analysis of PAR-2 responses and Tukey's t-tests to compare responses between groups.

 
Although it has been presumed that PMA-induced PAR-2 desensitization is due to activation of PKC, there may be other pathways activated by phorbol esters. To test this hypothesis, we examined the sensitivity of the PMA effect to inhibition by a broad-spectrum PKC inhibitor, GF109203X (GFX). As shown in Fig. 3, the PMA-induced suppression of Ca2+ mobilization observed in PAR-2GFP-transfected MEFwt or in untransfected MDA MB-468 cells was rescued by inhibition of PKC with GFX. Interestingly, homologous desensitization was not completely blocked by GFX (P. Kumar and K. DeFea, unpublished observations), suggesting the involvement of additional kinases in the regulation of PAR-2 desensitization.


Figure 3
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  		Fig. 3. PMA-induced desensitization is rescued by treatment with a broad-spectrum PKC inhibitor (GFX). MEFwt (A–D) or MDA MB-468 cells (E–H) were treated with vehicle (A and E), 300 nM GFX alone (B and F), 1 µM PMA alone (C and G), or PMA+GFX (D and H), and PAR-2-induced Ca2+ mobilization was determined in response to 50 nM trypsin, as described in Figs. 1 and 2.

 
Differential kinetics of PAR2 internalization in the absence of either beta-arrestin-1 or 2. To examine the possibility that beta-arrestins differentially regulate the endocytosis of PAR-2, we first measured internalization of endogenous PAR-2 in MEFwt, MEFbetaarrDKO, DKO+betaarr1, and DKO+betaarr2 by quantifying cell surface receptor levels using an antibody to the extracellular N terminus followed by fluorescence-activated cell scanning. The antibody to the N terminus recognizes an epitope that is partially removed by trypsin activation; thus, for this study and all internalization assays, a nonproteolytic agonist, 2fAP, was used. The kinetics of PAR2 internalization appeared to be the same, in response to trypsin, as assessed by confocal microscopy (data not shown). Cells were treated with 2fAP, for 0–60 min and internalization was determined by loss of surface fluorescence (Fig. 4). In MEFwt, a 55 ± 5% loss of surface fluorescence was observed as early as 5 min after agonist addition and was maintained for 60 min. In MEF DKO, no significant internalization was observed. Interestingly, in DKO+betaarr1 cells, a biphasic pattern of trafficking was observed. PAR-2 was rapidly internalized (by 58 ± 10% of control) within 5 min of receptor activation but by 15–30 min, the surface fluorescence values returned to baseline. By 60 min, surface fluorescence again decreased by 20 ± 5%. In DKO+betaarr2 cells, internalization occurred more slowly, with a significant decrease in surface fluorescence (60 ± 2%) observed after 30–60 min.


Figure 4
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  		Fig. 4. beta-arrestin-1 mediates early and beta-arrestin-2 mediates late PAR-2 internalization. MEFwt, MEF betaarrDKO, MEFDKO+betaarr1, and MEFDKO+betaarr2 cells were treated with PAR-2 agonist (100 nM 2fAP) for 0–60 min, and endogenous cell surface receptor levels were determined by immunoreactivity to an antibody directed against the extracellular N terminus using flow cytometry. Decreases in surface fluorescence relative to baseline reflect receptor internalization.

 
The differential kinetics of PAR2 internalization in MEFs was confirmed by confocal microscopy (Figs. 5 and 6). PAR2-GFP was transfected in each of the four MEF cell lines, and its trafficking and colocalization with recycling endosomal marker (transferrin, Fig. 5A), early endosomal marker (EEA1, Fig. 5B), and lysosomal marker (LAMP1, Fig. 6) was monitored. As has been reported by others, PAR2-GFP colocalized with EEA1 and transferrin within 5 min of activation in MEFwt and with LAMP1 after 60 min. Consistent with flow cytometry data, PAR2-GFP did not colocalize with EEA1 until 30 min of activation in DKO+betaarr2 cells, whereas in DKO+betaarr1 cells, this colocalization was observed within 2 min. Colocalization of PAR-2GFP with LAMP1 was observed in MEFwt and DKO+betaarr1, but not betaarrDKO or DKO+betaarr2 cells (Fig. 6A). Colocalization of endogenous PAR-2 with beta-arrestin-1 and LAMP-1 was also observed in MDA MB-468 cells after prolonged agonist exposure, while beta-arrestin-2 colocalized with PAR-2 but not with LAMP-1 (Fig. 6B). Furthermore, although beta-arrestins translocated to the membrane and colocalized with PAR-2GFP in MEFs within 2–5 min of PAR-2 activation, very little beta-arrestin-1 remained colocalized after 15 min (Fig. 7). In contrast, the majority of PAR-2/beta-arrestin-2 colocalization was observed after 30–60 min of receptor activation (Fig. 7). These data suggest that beta-arrestin-1 may mediate a lysosomal degradation of PAR-2; whereas beta-arrestin-2 might mediate other postinternalization fates.


Figure 5
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  		Fig. 5. Localization of PAR-2 with endosomal markers. A: MEFwt, MEFbetaarrDKO, MEFDKO+betaarr1, and MEFDKO+betaarr2, transiently transfected with PAR-2GFP, were bound with Cy5-transferrin (TF, a marker for constitutive endocytosis) on ice, then warmed to 37°C in the presence or absence of PAR-2 agonist (100 nM 2fAP) for 0–30 min. B: MEFs, transiently transfected with PAR2GFP were treated with 100 nM 2fAP for 2, 5 and 30 min, fixed, and costained with a marker of early endosomes (EEA1). Arrows indicate internalized receptor colocalized with either TF or EEA1. Insets to the left of each composite are x6 zooms of boxed regions.

 

Figure 6
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  		Fig. 6. Localization of PAR-2 with lysosomes. A: MEFwt, MEFbetaarrDKO, MEFDKO+betaarr1, and MEFDKO+betaarr2, transiently transfected with PAR-2GFP, were treated with 100 nM 2fAP for 0–60 min and were fixed and stained with a marker for degradative lysosomes (LAMP-1). Arrows indicate colocalized vesicles. Insets shown to the left and right of composites represent x5 zooms. B: MDA-MB-468 cells (which express high levels of endogenous PAR-2) transiently transfected with beta-arrestin-1 and 2-GFP were treated with 100 nM 2fAP for 2 h, and colocalization of PAR-2 and LAMP-1 with beta-arrestin-1 (top) or beta-arrestin-2 (bottom) was visualized by confocal microscopy.

 

Figure 7
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  		Fig. 7. Time course of beta-arrestin translocation and colocalization with PAR-2. CHO-PAR2 cells (stably expressing PAR-2-GFP) were transiently transfected with either Flag-beta-arrestin-1 (left) or Flag-beta-arrestin-2 (right) and treated with 100 nM 2fAP for 0–60 min. Note that both beta-arrestins colocalize with PAR-2 within 5 min, but beta-arrestin-2 remains colocalized longer. Arrows indicate colocalization.

 
Role of beta-arrestins in exocytosis of PAR2. A considerable portion of PAR-2 is stored in the Golgi following its translation, and agonist-induced trafficking of PAR-2 has been reported. This trafficking event was sensitive to brefeldin A (BFA) but not cyclohexamide, suggesting Golgi stores of PAR-2 are mobilized to the surface upon PAR-2 activation (15). Internalization assays revealed that some surface expression of PAR-2 returned after 30 min of agonist treatment in MEFwt and DKO+betaarr1; whereas, in DKO+betaarr2, the receptor appeared to remain internalized. To determine whether beta-arrestins were involved in the exocytosis of PAR-2, we examined agonist-induced trafficking of PAR-2-GFP after exposure to the Golgi-disrupting agent BFA. The appearance of PAR-2-GFP at the surface, after 30 min of 2fAP treatment, was abolished by BFA treatment in MEFDKO+betaarr1 (Fig. 8).


Figure 8
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  		Fig. 8. Disruption of Golgi apparatus with brefeldin A blocks PAR-2 reappearance at plasma membrane. MEFwt (A) and MEFDKO+betaarr1 (B) were treated with 100 nM 2fAP for 0–30 min in the presence and absence of 3 µg/ml brefeldin A (BFA). Note that the reappearance of PAR-2 at the plasma membrane in DKO+betaarr1 cells is blocked by BFA treatment.

 
Differential regulation of signaling molecules downstream by beta-arrestins 1 and 2. PAR2 has been shown to signal through beta-arrestins, leading to activation of some proteins (ERK1/2) and inhibition of others (phosphatidylinositol-3 knase) (13, 18, 43). Studies from other laboratories suggest that GPCRs can activate ERK1/2 by both G protein-dependent and independent pathways, the latter being beta-arrestin-dependent (37, 38, 44). Because the beta-arrestins differ temporally in their regulation of PAR-2 internalization, and previous studies suggest both beta-arrestins are necessary for full ERK1/2 activation, we investigated the specific contribution of each using the four MEF cell lines. In MEF wild-type cells, PAR-2 activation resulted in a biphasic ERK1/2 phosphorylation, with an early peak at 5 min and a later peak at 30–60 min (Fig. 9A). In DKO+betaArr1 cells, only the early increase at 5 min was observed, while in DKO+betaArr2 cells, we observed a slow rise in ERK1/2 phosphorylation at 5–60 min (Fig. 9, C and D). In MEF DKO cells, early ERK1/2 phosphorylation was reduced by 50%, and prolonged ERK1/2 phosphorylation at 30–60 min was abolished (Fig. 9B). PAR-2 has been reported to promote both membrane retention and nuclear translocation of activated ERK1/2, the former being associated with chemotaxis and the latter with proliferation (5, 9, 13, 17, 18, 29, 35). In mouse fibroblasts (NIH3T3) cells, beta-arrestin-1 appeared to prolong ERK1/2 activation for up to 90 min in isolated pseudopodia (17). Therefore, we examined whether beta-arrestins-1 and 2 differentially regulate membrane-associated ERK1/2. Subcellular fractionation of MEFs revealed that PAR-2 promotes a transient nuclear translocation of ERK1/2, which is prolonged in the absence of both beta-arrestins (Fig. 10, A and B). In contrast, membrane-associated phospho-ERK1/2 is abolished in the absence of beta-arrestins (Fig. 10B). Expression of either beta-arrestin-1 or beta-arrestin-2 alone abolished the prolonged nuclear ERK1/2 and resulted in the appearance of membrane-associated phospho-ERK. Furthermore, expression of beta-arrestin-2 appeared to specifically prolong membrane-associated ERK1/2 activation (Fig. 10, C and D).


Figure 9
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  		Fig. 9. beta-arrestin-1 mediates early and beta-arrestin-2 mediates late ERK1/2 activation. MEF wt (A), MEF betaarrDKO (B), DKO+betaarr1 (C) and DKO+betaarr2 (D) cells were treated with 50 nM trypsin for 0–60 min, or 20% serum (ser) for 5 min as a positive control, and lysates were analyzed by Western blot analysis for phospho (pERK) and total ERK (tERK). E: graph depicting normalized phospho-ERK levels as a fraction of baseline; values shown represent means ± SE from 4 independent experiments.

 

Figure 10
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  		Fig. 10. Subcellular fractionation of pERK in MEFs. MEFwt (A), betaarrDKO (B), DKO+betaarr1 (C), and DKO+betaarr2 (D), cells were treated with 50 nM trypsin for 0–90 min, after which cytosolic, nuclear, and plasma membrane fractions were analyzed for phospho-ERK by Western blot analysis. Shown are nuclear and plasma membrane fractions (left). Percentages of pERK in each fraction was calculated by densitometry and expressed as a bar graph to the right of each panel.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
beta-arrestins are known to play important roles in a variety of cellular processes, from desensitization of membrane receptors to facilitation of signal transduction pathways. In the case of PAR-2, studies have suggested that both beta-arrestins are required for efficient desensitization, internalization, and ERK1/2 activation (17, 18, 38), which suggested that they might have overlapping, as well as distinct roles in these processes. Here, we demonstrate that beta-arrestin-1 and 2 differ temporally in their regulation of PAR-2 desensitization, internalization, and ERK1/2 activation, suggesting it is their coordinated functions that are essential for PAR-2 signaling.

Role of beta-arrestins in G protein-uncoupling and PKC-mediated desensitization. The first event in receptor desensitization is the termination of the initial signal, usually accomplished through uncoupling of the receptor from its cognate G protein (10, 33). Receptor G protein uncoupling results in two phenomena: 1) The initial signaling event is terminated, and 2) the receptor becomes refractory to second agonist stimulation. In these studies, G{alpha}q coupling is measured by release of intracellular Ca2+ in response to the PAR-2 agonists, trypsin and 2fAP. In MEFs from beta-arrestin–/– mice, the duration of Ca2+ release is nearly tripled, consistent with ineffective uncoupling of PAR-2 from G{alpha}q. Expression of beta-arrestin-1, but not beta-arrestin-2, effectively restores signal termination, suggesting that beta-arrestin-1 mediates this initial uncoupling event. In many cases, desensitization is initiated upon phosphorylation of receptors by G protein-receptor kinases (GRKs) and subsequent binding of beta-arrestins. GRKs are activated in response to the same receptors that utilize them, reflecting a negative feedback loop designed to prevent constitutive receptor signaling. Although PAR-2 uses beta-arrestins for internalization (15), no role for GRKs in PAR-2 signaling has been demonstrated yet. In contrast, studies have revealed that activation of PKC promotes receptor desensitization and internalization, and mutation of putative PKC phosphorylation sites impairs these same processes (7, 13). Whether PKC itself or another downstream kinase directly phosphorylates PAR-2 remains unknown, but here, we demonstrate that beta-arrestins are required for PKC-mediated desensitization. Pretreatment with PMA did not block PAR-2-promoted mobilization of internal Ca2+ in MEFs from beta-arrestin–/– mice or in wild-type cells after treatment with a PKC inhibitor. After expression of either beta-arrestin-1 or 2 in the MEF betaarrDKO cells, PMA once again inhibited PAR-2 mediated Ca2+ mobilization, suggesting their functions are redundant at this step in PAR-2 desensitization. Surprisingly, inhibition of PKC did not block homologous desensitization (P. Kumar and K. DeFea, unpublished observations), suggesting that other kinases (perhaps GRK) mediate agonist-induced G-protein uncoupling. Thus heterologous desensitization by PKC may reflect a contribution of other cellular pathways in the regulation of PAR-2 activation.

Role of beta-arrestins in PAR-2 trafficking. In MEFbetaarr-DKO cells, agonist-induced PAR-2 internalization was virtually abolished. Interestingly, expression of beta-arrestin-1 alone restored early, while expression of beta-arrestin-2 alone restored prolonged receptor internalization. Taken together, with the apparent ability of beta-arrestin-1 to mediate rapid termination of PAR-2 induced Ca2+ mobilization, one might presume a temporal difference in the recruitment of each beta-arrestin to the membrane. Such a distinction would have to occur within seconds after receptor activation, as both beta-arrestin-1 and 2 colocalized with PAR-2 within 5 min of its activation. Whether both beta-arrestins can simultaneously interact with PAR-2 or whether each regulates a distinct receptor pool remains to be determined. Colocalization with early endosomal markers was observed with expression of either beta-arrestin-1 or 2, but targeting to lysosomes, as evidenced by colocalization of PAR-2 with LAMP-1, was only rescued by expression of beta-arrestin-1. Consistent with this finding, in cells expressing high levels of endogenous PAR-2, LAMP-1 colocalized with beta-arrestin-1 after prolonged agonist exposure, but not with beta-arrestin-2, despite the fact that PAR-2 colocalized with both. Whether this result reflects a distinct fate of beta-arrestin-2-bound receptor remains to be determined. Reports by others of beta-arrestin-2 ubiquitination and ubiquitin-mediated degradation of PAR-2 raise the possibility that beta-arrestin-2 might mediate ubiquitin-directed PAR-2 degradation, while beta-arrestin-1 mediates lysosomal degradation (19, 36, 41). Another possibility is that beta-arrestin-2-associated receptors are involved in prolonged ERK1/2 activation (discussed in Role of beta-arrestins in ERK1/2 activation).

Another surprising observation was the fact that restoration of surface PAR-2 was enhanced by expression of beta-arrestin-1 alone, and this was sensitive to the Golgi-disrupting drug, BFA. One explanation for this observation is that beta-arrestin-1 not only mediates early endocytosis and degradation of PAR-2 but also facilitates trafficking of receptor from Golgi stores to the membrane in response to agonist stimulation. There is precedent for such a hypothesis, as beta-arrestin-1-facilitated anterograde trafficking was reported for Ral-GDS (6), and PAR-2 has previously been shown to exhibit BFA-sensitive agonist-induced exocytosis (15, 34). Alternatively, beta-arrestin-1 might mediate rapid endocytosis and degradation of PAR-2, which is replaced by constitutive exocytosis.

Role of beta-arrestins in ERK1/2 activation. It has previously been shown that PAR-2-evoked ERK1/2 activation requires both beta-arrestins, although there may be a temporal difference in their involvement (13, 18, 38). Here, we provide evidence that PAR-2-evoked ERK1/2 activation involves at least three separate pathways: an early beta-arrestin-1-dependent phase (2–5 min), a prolonged beta-arrestin-2-dependent phase, and a third beta-arrestin-independent component. The temporal involvement of each beta-arrestin is consistent with their apparent involvement in receptor internalization; moreover, the two beta-arrestins do not appear to require each other for regulation of ERK1/2 activity. In previous studies, a mutant receptor lacking two putative C-terminal PKC phosphorylation sites was able to exert prolonged Ca2+ mobilization, leading to activation of Src-family kinases, nuclear ERK1/2 activation and proliferation; whereas the wild-type receptor primarily utilized the beta-arrestin-dependent pathway, resulting in cytosolic sequestration of activated ERK1/2. In these studies, PAR-2 evoked nuclear translocation of active ERK1/2 appeared to be unaffected in MEF-betaarrDKO; expression of beta-arrestin-1 or -2 increased membrane-associated ERK1/2. These data further suggest that the two beta-arrestins may regulate distinct pools of activated ERK1/2, just as they appear to regulate distinct pools of receptor.

There are a growing number of receptors that appear to exert beta-arrestin-dependent, G protein-independent ERK1/2 activation. In some cases, beta-arrestins stably associate with ERK1/2 (e.g., downstream of PAR-2), leading to its sequestration away from the nucleus, whereas in other cases, they facilitate its nuclear transport (12, 13, 39, 40). In the case of PAR-2, there is evidence to suggest that residues in the receptor C terminus may define the duration of ERK1/2 association with beta-arrestin, the specificity of beta-arrestin association, and the mechanism of activation (13, 35, 38). Tissue and cell-type specific differences in beta-arrestin levels, may then result in differences in Ca2+ signal duration and receptor internalization, as well as tip the scales toward nuclear or cytosolic/membrane ERK1/2, ultimately resulting in distinct cellular outcomes of PAR-2 activation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by R01GM066151 to K. A. DeFea.


   ACKNOWLEDGMENTS
 
We are thankful to Dr. Robert Lefkowitz (Duke University Medical Center) for MEFs from beta-arrestin–/– mice and GFP-tagged beta-arrestin constructs, to David Carter (UCR Integrative Genome Center Microscopy Suite) for assistance with confocal microscopy, Youly Ly for technical assistance, and Dr. Christian Lytle for assistance with single-cell Ca2+ mobilization assays.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. DeFea, Div. of Biomedical Sciences, 1620 Computer Statistics Bldg., Univ. of California, Riverside, CA 92521 (e-mail: kathryn.defea{at}ucr.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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