Neutrophil Elastase Acts as a Biased Agonist forProteinase-activated Receptor-2 (PAR2)*□S

Received for publication, November 19, 2010, and in revised form, May 13, 2011 Published, JBC Papers in Press, May 16, 2011, DOI 10.1074/jbc.M110.201988

Rithwik Ramachandran‡�1, Koichiro Mihara‡�, Hyunjae Chung‡�2, Bernard Renaux‡�, Chang S. Lau§,Daniel A. Muruve�, Kathryn A. DeFea§, Michel Bouvier¶, and Morley D. Hollenberg‡�

From the ‡Department of Physiology and Pharmacology and �Department of Medicine, Faculty of Medicine, University of Calgary,Calgary, Alberta T2N 4N1, Canada, the §Division of Biomedical Sciences and Biochemistry and Molecular Biology, University ofCalifornia, Riverside, California 92521, and the ¶Department of Biochemistry, Institute for Research in Immunology and Cancer,Groupe de Recherche Universitaire sur le Medicament, Universite de Montreal, CP 6128 Succursale Centre-Ville,Montreal, Quebec H3C 3J7, Canada

Human neutrophil proteinases (elastase, proteinase-3, andcathepsin-G) are released at sites of acute inflammation. Wehypothesized that these inflammation-associated proteinasescan affect cell signaling by targeting proteinase-activated recep-tor-2 (PAR2). The PAR family of G protein-coupled receptors istriggered by a unique mechanism involving the proteolyticunmasking of an N-terminal self-activating tethered ligand(TL). Proteinases can either activate PAR signaling by unmask-ing the TL sequence or disarm the receptor for subsequentenzyme activation by cleaving downstream from the TL se-quence.We found that noneofneutrophil elastase, cathepsin-G,and proteinase-3 can activate Gq-coupled PAR2 calcium signal-ing; but all of these proteinases can disarm PAR2, releasing theN-terminal TL sequence, thereby preventing Gq-coupled PAR2signaling by trypsin. Interestingly, elastase (but neither cathep-sin-G nor proteinase-3) causes a TL-independent PAR2-medi-ated activation of MAPK that, unlike the canonical trypsinactivation, does not involve either receptor internalization orrecruitment of �-arrestin. Cleavage of synthetic peptidesderived from the extracellularN terminus of PAR2, downstreamof the TL sequence, demonstrated distinct proteolytic sites forall three neutrophil-derived enzymes. We conclude that ininflammation, neutrophil proteinases can modulate PAR2 sig-naling by preventing/disarming the Gq/calcium signal pathwayand, via elastase, can selectively activate thep44/42MAPKpath-way. Our data illustrate a new mode of PAR regulation thatinvolves biased PAR2 signaling by neutrophil elastase and a dis-arming/silencing effect of cathepsin-G and proteinase-3.

Neutrophil infiltration represents a rapid innate immunedefense mechanism that accompanies acute inflammation.

Although one main neutrophil function is to target invadingmicroorganisms and to respond to damaged tissue, there aremany host cell responses triggered by the released neutrophilserine proteinases. In inflammation, apart from their bacteri-cidal actions, the serine proteinases trigger a variety of effectsranging from cytokine, kinin, and growth factor generation tothe clustering of integrins (1). PARs3 belong to a novel four-member family ofG-protein-coupled receptors that are proteo-lytically activated by the unmasking of an N-terminal tetheredligand (TL) sequence by serine proteinases (2–4). Of note,many proteinases can silence/disarm PARs by cleaving down-streamof theTL sequence, so as to prevent further activation byproteinases, but not by synthetic PAR-activating peptides withsequences derived from the TL (2, 5). In previous work, wefound that neutrophil elastase (NE) and cathepsin-G (CG) canblock Gq-mediated calcium signaling by trypsin-activated pro-teinase-activated receptor-2 (PAR2), presumably by removingthe enzyme-targeted tethered ligand sequence (6, 7). PAR2 acti-vation by serine proteinases is widely considered to cause aproinflammatory response in various pathological conditions,in part via a neurogenic mechanism, and thus its disarming byneutrophil proteinases could be considered as an anti-inflam-matory effect. There are also intriguing data that assign a pro-tective function to PAR2 activation in certain inflammatoryconditions (8–11). The mechanism(s) for this potentiallybiphasic action of PAR2 is not known, but it may relate to theability of PAR2 to exhibit biased signaling either via a Gq-cou-pled calcium signal or via Gi/G12/13-coupled p44/42MAPK sig-nals (12). Our recent report showed that proteolyticallyrevealed mutated rat TL sequences and one of the PAR2-acti-vating peptides we tested were able to trigger such biased ago-nism, activating MAPK but not calcium signaling pathways,without either receptor internalization or the recruitment ofarrestins (12). Evidence for biased signaling by PAR2 also comesfrom the observation that a novel peptidomimetic PAR2 antag-onist could inhibit intracellular calcium release induced by aPAR2 agonist but failed to inhibit ERK signaling (13). It is, how-ever, not known if endogenous agonists can trigger biased sig-

* This work was supported by grants from the Canadian Institutes of HealthResearch (to M. D. H. and M. B.). This work was also supported in part byNational Institutes of Health Grant RO1GM066151 (to K. D.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table 1 and Figs. S1–S3.

1 An Alberta Innovates Health Solutions/Alberta Heritage Foundation forMedical Research (AI-HS/AHFMR) postdoctoral fellow. To whom corre-spondence should be addressed: Dept. of Physiology and Pharmacology,Faculty of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary,Alberta T2N4N1, Canada. Tel.: 403-220-3663; Fax: 403-270-0979; E-mail:rramacha@ucalgary.ca.

2 Supported by a CHO/FoMD University of Alberta Emerging Teams Grantgraduate scholarship.

3 The abbreviations used are: PAR, proteinase-activated receptor; TL, teth-ered ligand; NE, neutrophil elastase; CG, cathepsin-G; PR3, proteinase-3;HPT, human proximal tubular; MEF, mouse embryo fibroblast; BRET, biolu-minescence resonance energy transfer; BAB, biarsenical fluorochromebinding domain; AMC, 7-amido-4-methylcoumarin.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 28, pp. 24638 –24648, July 15, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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naling through PAR2. In trying to uncover if such biased signal-ing might occur in vivo, we hypothesized that neutrophilproteinases that can cleave the PAR2 N terminus but do nottrigger PAR2 calcium signaling might nonetheless activateMAPK to act as endogenous biased agonists for PAR2.We showhere that neutrophil proteinases (NE, CG, and proteinase-3(PR3)) can all disarm PAR2-dependent calcium responses totrypsin, whereas NE (but neither CG nor PR3) concomitantlyactivatesMAPK signaling.We show further that all three neu-trophil proteinases can target the PAR2 N terminus toremove the TL but via cleavage at distinct cleavage sites.We propose that under inflammatory conditions, this novelTL-independent signaling via PAR2, biased toward theMAPK pathway, may underlie differential PAR2-dependentresponses in inflammation.

EXPERIMENTAL PROCEDURES

Chemicals and Other Reagents—Culture medium (DMEMand DMEM/F-12) and fetal bovine serum (FBS) were fromInvitrogen. Fluo-4/AM no wash calcium indicator dye and theReASH fluorescent probe were from Invitrogen. Fugene-6transfection reagent was obtained fromRocheApplied Science.Calcium ionophore A23187, porcine trypsin (catalogue no.T-7418; �14,900 units/mg), collagen type IV from human pla-centa, insulin/transferrin/sodium selenite, hydrocortisone,5-triiodo-L-thyronine sodium salt, PGE-1, and EGF were fromSigma. A maximum specific activity of 20,000 units/mg wasused to calculate the approximate molar concentration of tryp-sin in the incubation medium (1 unit/ml, �2 nM). The neutro-phil enzymes, neutrophil elastase, cathepsin-G, and proteinase3, purified from human sputum, were obtained from ElastinProducts (Owensville, MO) with specific activities of 875, 3.1,and 95 units/mg, respectively. No trace trypsin-like activity wasdetectable in the enzymes as confirmed by us with a 96-wellplate-based enzyme activity assay using the fluorogenic sub-strate, t-butoxycarbonyl-QAR-AMC (Bachem, Torrence, CA).The maximum concentrations of enzymes in our experimentswere 1 �M at 3 units/ml for NE and PR3 and 4 �M at 300 milli-units/ml for CG. TheNE inhibitor, fragment of elafin, was fromAnaspec (Fremont, CA). NE activity was measured using thefluorogenic substrate methyl-O-succinyl-Ala-Ala-Pro-Val-AMC (elastase substrate V, EMD Chemicals, Gibbstown, NJ).The PAR-activating peptide, SLIGRL-NH2, and the rat PAR2-derived sequence ITGKGAPVEPGFSVDEFSASVLTGKLT(D27) as well as the TL-containing sequences (TL sequenceunderlined) for human PAR1 (NATLDPRSFLLRNPNDKYE),PAR2 (GTNRSSKGKSLIGKVDGTSHV), andPAR4 (GDDSTP-SILPAPRGYPGQV) used as enzyme substrates were synthe-sized by the Faculty ofMedicine Peptide Synthesis facility at theUniversity of Calgarywith purity verified by amino acid analysisand mass spectral analysis.Cell Culture—KNRK and HEK cells were cultured as

described previously (12). Briefly, cells were grown to conflu-ence in DMEM supplemented with 10% FBS and 1% antibiotic/antimycotic. Cells that were transfected with either the ratPAR2-encoding pCDNA3.1 vector or the empty pCDNA3.1vector were grown in DMEM supplemented with 600 �g/mlG418. Confluent cultures were dissociated in PBS containing 1

mM EDTA and subcultured as appropriate. Mouse embryonicfibroblasts from wild type (MEFwt) and �-arrestin-1/2 doubleknock-out animals (MEF�arrDKO) were a gift fromDr. RobertLefkowitz and have been described previously (14, 15).Primary human proximal tubular (HPT) cells were isolated

as described before (16). Briefly, normal renal cortical tissueobtained from an adult kidney undergoing nephrectomy wasdissected, minced, digested with collagenase IV, and passedthrough a 75-�m mesh. Filtered cells were washed in Hanks’balanced salt solution and resuspended in hormonally definedmedium and plated in a Petri dish. After 30 min, the non-ad-herent HPT cells were transferred to a collagen-coated plateand cultured in DMEM/F-12 (Invitrogen) supplemented with1% FBS, 1% penicillin/streptomycin, 125 ng/ml prostaglandinE1, 25 ng/ml epidermal growth factor, 1.8 �g/ml L-thyroxine,3.38 ng/ml hydrocortisone, and 2.5 mg/ml insulin/transferrin/sodium selenite supplement. Experiments involving the use ofhuman tissue were approved by the Conjoint Health ResearchEthics Committee at the University of Calgary.Cloning and Transfection—The plasmid encoding the rat

PAR2 receptor (wt-rPAR2) was constructed as described previ-ously (17). Permanent KNRK cell lines expressing PAR2 andpCDNA3.1 have been described previously (17). HEK-293 cells,used for the PAR2-biarsenical fluorochrome binding domain(BAB) construct cleavage experiments and the biolumines-cence resonance energy transfer (BRET) experiments, weretransiently transfected with the pcDNA3.1� vector encodingthe human PAR2 receptor with the N-terminal BAB tag or thepcDNA3.1 vector encoding the human PAR2 gene with a c-ter-minal YFP tag, using the Fugene 6 (Roche Applied Science)transfection reagent (12). Transiently transfected cells wereassayed 48 h post-transfection.Measurement of Trypsin and NE Activity—NE activity was

measured using the substrate methyl-O-succinyl-Ala-Ala-Pro-Val-AMC (elastase substrate V, EMD Chemicals). 50 �l of 200milliunits/ml elastase in Hanks’ balanced salt solution contain-ing 10 mM HEPES, pH 7.0, 1.5 mM MgCl2, 1.5 mM CaCl2, and0.2%Nonidet P-40was incubatedwith 50�l of 200�Melastase-specific substrate methyl-O-succinyl-Ala-Ala-Pro-Val-AMC(elastase substrate V, EMD Chemicals). The fluorescence fromthe free AMC as an index of proteolytic activity was measuredevery 10 min for 60 min using a Victor X4 fluorescent platereader (PerkinElmer Life Sciences). In order to confirm inhibi-tion of NE by elafin, NE and elafin were preincubated for 5 minat room temperature prior to measuring NE activity asdescribed above. Trypsin activity was measured using the sub-strate t-butoxycarbonyl-QAR-AMC with similar conditions asfor NE. The effect of neutrophil elastase on trypsin activity wasmeasured by preincubating the two enzymes for 5 min at 37 °Cprior to measuring trypsin activity.Calcium Signaling—KNRK cells orHPT cells were incubated

for 30 min with Fluo-4/AM no wash calcium indicator andassayed for calcium signaling responses as described previously(12) on a victor X4 fluorescent plate reader (PerkinElmer LifeSciences) or an Aminco Bowman fluorescence spectrophoto-meter (Thermo Life Science). Receptor disarming was moni-tored essentially as described previously (18), with minor mod-ifications. Briefly, PAR2-expressing KNRK cells or HPT cells

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were first exposed or not to 0.3–3 units/ml of the three neutro-phil proteinases for 20 min before assaying responses toincreasing concentrations of trypsin or SLIGRL-NH2.p44/42 MAPK Signaling—Western blot detection of p44/42

MAPK was performed essentially as described (12). In brief,KNRK cells that had been transfected with a PAR2-expressingconstruct (PAR2-KNRK) or empty vector (pcDNA3-KNRK)were placed in DMEM with no serum for 12 h and stimulatedwith either trypsin, SLIGRL-NH2, NE, CG, or PR3 for 10 min.Cells were then placed on ice, and agonists were removed priorto lysis with cold lysis buffer. Protein samples were boiled for 10min in Laemmli buffer and resolved on SDS-polyacrylamidegels. Activation of p44/42 MAPK was monitored by immuno-blotting with phospho-p44/42 MAPK-specific antibodies andvisualizing on an Eastman Kodak Co. Imagestation, and theincreases in p44/42 MAPK phosphorylation were quantifiedrelative to the total p44/42 MAPK or �-actin signal detected inthe same samples. Densitometry analysis of multiple Westernblot images was done to quantify the percentage increase overbase line of p44/42 MAPK activation by the different protei-nases. MAPK signaling in HPT cells was assessed as describedabove for KNRK cells. For p44/42MAPK studies inMEFs, cellswere treated as described for KNRK cells except that after incu-bation with agonists, cells were washed with ice-cold PBS andlysed directly in 1� Laemmli sample buffer. Phospho-p44/42MAPK and total p44/42 MAPK antibodies were visualized onWestern blots with Alexa 680 and IR800-coupled secondaryantibodies using the LICOR Odyssey Imaging system.Measurement of PAR2 “Disarming” byNeutrophil Proteinases

Using Biarsenical Fluorochrome Binding Motif-tagged PAR2—The BAB, containing a tetracysteinemotif (19), AFLNCCPGC-CMEP, was inserted by introducing corresponding oligonu-cleotides just downstream of the signal peptide of human PAR2(Gln27). The PAR2 DNA constructs containing the N-terminalBAB domain and a C-terminal YFP fluorochrome tag werecloned in pCDNA3.1� at the restriction enzyme sites betweenEcoRI and XbaI (PAR2-BAB-YFP). HEK-293 cells obtainedfrom ATCC (Manassas, VA) were transfected with the PAR2-BAB-YFP construct with the FuGENE6 transfection reagent(Roche Applied Bioscience). The expression level of theexpressed receptor was quantified using a fluorescence platereader (Victor 4X, PerkinElmer Life Sciences), and confocalmicroscopy was used to monitor the receptor location by visu-alizing the receptor YFP tag. To detect proteolytic release of theN-terminal PAR2 domain, PAR2-BAB-YFP cells were labeledwith a 0.2 �M concentration of the biarsenical fluorochromeReAsH (using the TC-TC-ReAsH II in-cell tetracysteine tagdetection kit (Invitrogen)) for 30 min followed by washing withthe included BAL buffer. Labeled cells were then incubatedwith proteinases for 30 min in HEPES-buffered saline, pH 7.4,containing 1.5mMCaCl2. Supernatants were removed from thecells and briefly centrifuged, and fluorescence measurementswere done using a Victor X4 fluorescent plate reader, with anexcitation wavelength of 540 nm and an emission wavelengthmonitored at 615 nm to detect release of the peptide containingthe receptor BAB tetracysteine motif. Fluorescence readingswere also obtained from the cell monolayer to detect loss or notof the BAB tetracysteine motif from the cell surface.

Neutrophil Proteinase Cleavage of Synthetic PAR2-derivedPeptides—Cleavage of the PAR2-derived synthetic peptidesspanning the TL regions (human PAR sequences; NATLDPRS-FLLRNPNDKYE (PAR1), GTNRSSKGKSLIGKVDGTSHV(PAR2), and GDDSTPSILPAPRGYPGQV (PAR4)) or en-compassing the region downstream of the PAR2 TL region(rat PAR2 sequence ITGKGAPVEPGFSVDEFSASVLTGKLT,highly homologous with the comparable human sequence,VTGKGVTVETVFSVDEFSASVLTGKLT) was done as de-scribed previously for trypsin IV and tissue kallikreins (20, 21).Peptides (100 �M in a total volume of 150 �l) were incubatedwith neutrophil proteinases (0.7 unit/ml) for 15 min at 37 °C.Reactions were stopped by adding 150 �l of ice-cold 0.1% tri-fluoroacetic acid (TFA) in water. Samples were fractionatedby reverse-phase high performance liquid chromatography(5–40% acetonitrile gradient in 0.1% TFA over 30 min at a flowrate of 1ml/min), and eluted peptideswere analyzed byMALDImass spectrometry. Neutrophil proteinase cleavage of peptidesspanning the TL regions of PAR1 (NATLDPRSFLLRNPND-KYE) and PAR4 (GDDSTPSILPAPRGYPGQV) were alsoassessed as described above for the PAR2 peptides.BRET-based Detection of �-Arrestin-1 Interaction with PAR2—

PAR2 was conjugated with YFP at the C terminus (PAR2-YFP),and BRET to Renilla luciferase-tagged �-arrestin-1 (Rluc-�-arr1) was determined essentially as described previously (22).HEK-293 cells were used instead of the KNRK cell line, becausetheHEK cells provide for amuch greater transfection efficiencythat is required for the BRET studies. In brief, HEK-293 cellswere transiently transfected with 1 �g of PAR2-YFP constructalong with 0.1 �g of the Rluc-�-arr1 construct. Cells wereplated in white 96-well culture plates (PerkinElmer Life Sci-ences), and interactions between the receptors and�-arrestin-1were detected by measuring BRET at timed intervals over 60min following the addition of 5 �M coelenterazine (NanolightTechnology, Pinetop, AZ) on a Mithras fluorescence platereader (Berthold, Mandel Scientific (Guelph, Canada)) in lumi-nescence mode using the appropriate filters.Monitoring PAR2 Internalization—Using the approach

described in more detail elsewhere (12), HEK cells were platedon glass bottom Petri dishes (MatTek Corp., Ashland,MA) andtransiently transfected with 1 �g of PAR2-YFP construct. After48 h, themedium in the Petri dishwas replacedwith serum-freemedium, and the cells were treated with trypsin (10 nM), SLI-GRL-NH2 (10�M), or NE (3 units/ml) for 30min at 37 °C. Afterfixing cells with 4% formaldehyde, the cellular localization ofthe receptor tag YFP signal was detected using an OlympusFV1000 confocal system on an Olympus IX70microscope withthe Fluoview system software. Internalization of cell surfacereceptor was quantified morphometrically by counting thenumber of intracellular fluorescent speckles per cell in theimages, indicative of receptor internalization to endocytic ves-icles. Speckles in all cells were counted in a randomly selectedrepresentative �40 image. The average number of speckles percell was calculated, and the observation was repeated for com-parable fields for cells observed in three independently con-ducted experiments.

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RESULTS

Neutrophil Proteinases Target Distinct Cleavage Sites andDisarm PAR2 by Cleaving the Receptor N Terminus Down-stream of the TL Sequence—As we anticipated from our previ-ous work (6, 7) and as shown in Fig. 1A (representative data forNE) and Fig. 1B, pretreatment of PAR2-expressing cells withNE, CG, or PR3 abrogated subsequent trypsin-mediated cal-cium signaling, without affecting the response to the PAR-acti-vating peptide, SLIGRL-NH2. The abrogation of the ability oftrypsin to activate PAR2 was not due to neutrophil proteinasedigestion of trypsin itself as confirmed by experiments wherepreincubating trypsin with NE (as opposed to its target cells)did not affect either its ability to cause calcium signaling or tocleave a fluorogenic substrate (supplemental Fig. S2). This lossof PAR2 sensitivity to trypsin activation upon pretreating cellswith the neutrophil proteinases can be explained by the abilityof all three enzymes to cleave and release the N-terminaldomain of PAR2 from the cell surface (Fig. 2), making the TLunavailable for trypsin-mediated unmasking. This N-terminalcleavage was verified by detection of the fluorescently labeledreceptor N-terminal fragment containing a biarsenical fluoro-chrome binding motif in the cell supernatant after enzymetreatment. To assess similarities or differences in the cleav-age of the PAR2 N-terminal extracellular sequence by the threeneutrophil proteinases, we used a synthetic 27-mer peptidesubstrate representing the rat PAR2 sequence downstream ofthe TL and just C-terminal to a series of three prolines thatwould segregate this sequence from the upstream TL moiety(Fig. 3).HPLC separation andmass spectral identification of thecleavage products showed that each of the enzymes cleaved at a

distinct site in this peptide with predominant cleavageobserved: at Ser68-Val69 for NE, at Phe65-Ser66 for CG, and atVal62-Asp63 for PR3 (Fig. 3).In contrast with the cleavage of the PAR2 27-mer that repre-

sents a PAR2 sequence downstream from the TL, we failed tosee any cleavage of the peptide corresponding to the humanPAR2 N-terminal region spanning the TL cleavage activationsite, GTNRSSKGK2SLIGKVDGTSHV (where the downwardarrow denotes the cleavage site that unmasks the TL) (data notshown). These studies indicated that the neutrophil-derivedproteinases are unable to reveal the PAR2-activating TL andagree completely with previous proteomic work demonstratingthe inability of neutrophil proteinases to cleave sequences rep-resenting the TL of PAR2 (23). Thus, neutrophil enzymes dis-arm PAR2 by removing the N-terminal TL receptor-activatingsequence, whereas the retention of calcium signaling in re-sponse to SLIGRL-NH2 after treatment with neutrophil protei-nases showed that the extracellular receptor loops involved insignaling remained functional.NE Activates PAR2-dependent p44/42 MAPK Signaling,

whereas CG and PR3 Do Not—Because we previously observedthat PAR2 can selectively couple to the p44/42 MAPK pathwaywithout triggering calcium release (12), we wondered if the neu-trophil proteinases might act as endogenous biased agonists atPAR2, selectivelyactivatingMAPKbutnotcalciumtransients.Fol-lowing activation of KNRK cells stably expressing rat-PAR2(KNRK-PAR2)withNE for10min (butnotwith eitherCGorPR3)we indeedobserveda robustp44/42MAPKsignalingwith anEC50for NE of about 0.7 unit/ml (Fig. 4). This response was notobserved in empty vector-transfected KNRK cells (Fig. 4C).

FIGURE 1. Neutrophil proteinases cleave PAR2 and disarm PAR2-coupledcalcium signaling. KNRK cells expressing PAR2 were incubated with theFluo-4 no wash calcium indicator for 30 min, and trypsin-stimulated calciumsignaling was monitored in cells that were preincubated or not with 3units/ml neutrophil-derived proteinases for 20 min. A, representative traceshowing the lack of NE stimulated calcium signaling in PAR2-KNRK cells andthe subsequent inability of trypsin to stimulate calcium signaling in thesecells (// indicates 20 min). The PAR2-AP SLIGRL-NH2 is still able to stimulatecalcium signaling in cells that are disarmed by NE. B, concentration-responsecurve for trypsin-stimulated calcium signaling in PAR2-expressing KNKR cells(open squares) and NE-, CG-, and PR3-pretreated PAR2-expressing KNRK cells.Error bars, S.E.

FIGURE 2. Neutrophil derived proteinases cleave the PAR2 N terminus.Assessment by measuring release of an engineered tetracysteine arsenitebinding motif. HEK-293 cells were transfected with the PAR2 DNA constructscontaining the N-terminal BAB domain and a C-terminal YFP fluorochrometag (PAR2-BAB-YFP). To detect proteolytic release of the N-terminal PAR2domain, cells were labeled with a 0.2 �M concentration of the biarsenicalfluorochrome ReAsH for 30 min followed by washing with BAL buffer. Labeledcells were then incubated with proteinases for 30 min in HEPES-bufferedsaline, pH 7.4, containing 1.5 mM CaCl2. Supernatants were removed from thecells and briefly centrifuged, and fluorescence was measured using a VictorX4 fluorescent plate reader, with an excitation wavelength of 540 nm and anemission wavelength monitored at 615 nm. Fluorescence readings wereobtained from the culture supernatants to detect release of the peptide con-taining the receptor BAB tetracysteine motif and from the cell monolayer todetect loss or not of the BAB tetracysteine motif from the cell surface (mean �S.E. (error bars), n � 3). *, significant increase in detectable released fluores-cence in assay buffer compared with untreated sample (NT); #, significantdecrease in detectable cell surface fluorescence compared with untreatedsample (p � 0.05).

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Despite being able to cleave the PAR2N terminus to prevent tryp-sin signaling, neither CG nor PR3 was able to activateMAPK sig-naling in PAR2-transfected KNRK cells (Fig. 4A).

Given that short synthetic peptides corresponding to thetrypsin-revealed N terminus of PAR2 can activate the receptor,we synthesized peptides derived from the N-terminal sequence

FIGURE 3. Determination of sites in the PAR2 N terminus downstream of the TL that are cleaved by neutrophil proteinases. The rat PAR2 N terminus-derived peptide was incubated with the neutrophil proteinases for 20 min, and the samples were fractionated by HPLC and identified by MALDI MS. A, repre-sentative HPLC tracings showing relative abundance of different proteinase cleavage products. The parent peptide is underlined, and the major peak detectedis shown in boldface type. B, schematic showing the PAR2 N terminus with the arrows showing the major sites of cleavage of the 27-mer PAR2-derived peptideby the three neutrophil proteinases and by trypsin. D27, downstream peptide 27 mer.

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of PAR2 exposed by CG (SASVLTGKLTTVFL-NH2) and NE(VLTGKLTTVFL-NH2) (Fig. 3). In contrast with the trypsin-revealed TL-derived peptide (SLIGRL-NH2), the NE/CG-re-vealed TL peptides (10–100 �M) were unable to trigger either acalcium signal orMAPK signal in PAR2-transfectedKNRKcells(supplemental Fig. S1).NEDisarms Calcium Signaling and ActivatedMAPK Signaling

in HPT Cells That Endogenously Express PAR2—In order to con-firm the existence of NE-dependent biased signaling in cells thatendogenously express PAR2, we have examined the effect of NE

on calcium and MAPK signaling in HPT cells that naturallyexpress high levels of PAR2 (24). Treatment of HPT cells withNEdidnot stimulate a calciumsignal but significantly attenuated sub-sequent trypsin-stimulated calcium transients (Fig. 5,A andB). Incontrast, treatment of HPT cells with NE for 5 min stimulatedrobust p44/42MAPK signaling that declined but remained higherthan base line at 15 and 30min post-treatment (Fig. 5,C andD).Rho-kinase Inhibitors and Elafin Block NE-activated PAR2-

dependent p44/42 MAPK Signaling—The NE-stimulatedMAPK activation was attenuated by Rho-kinase inhibitors (Fig.

FIGURE 4. NE activates PAR2-dependent p44/42 MAPK signaling, whereas CG and PR3 do not. Rat PAR2-expressing KNRK cells or empty vector-expressingcells (pcDNA3-KNRK) were serum-starved and stimulated with either trypsin, SLIGRL-NH2, NE, CG, or PR3 for 10 min. Samples were resolved on SDS-polyacryl-amide gels, and activation of p44/42 MAPK was monitored by immunoblotting with phospho-p44/42-specific antibodies. A, representative image showing activationof p44/42 MAPK by NE but not by CG and PR3 in rPAR2-KNRK cells. B, histogram showing densitometry analysis of multiple scanned Western blot images to quantifythe percentage increase over base line of p44/42 activation by the different proteinases in rPAR2-KNRK cells. C, representative image showing no activation of p44/42MAPK by NE in pcDNA3-KNRK cells. D, histogram showing densitometry analysis of multiple scanned Western blot images to quantify the percentage increase overbase line of p44/42 activation by the different proteinases in pcDNA3-KNRK cells. E, representative image showing activation of p44/42 MAPK in PAR2-KNRK cells inresponse to different concentrations (0.75–3 units/ml) of NE. F, histogram showing densitometry analysis of multiple scanned Western blot images to quantify thepercentage increase over base line of p44/42 activation by increasing concentrations of NE. Increases in p44/42 MAPK phosphorylation were quantified relative to the�-actin or total p44/42 signal detected in the same samples (mean � S.E. (error bars), n � 3; *, p � 0.05). NT, no treatment.

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6). In order to confirm the specificity of NE-stimulated MAPKactivation, we monitored the ability of NE to activate MAPKfollowing incubation with the specific NE inhibitor elafin (sup-plemental Fig. S3). We found that the NE-stimulated MAPKactivation was completely inhibited by 10 �M elafin and thatthis inhibition was dependent on the elafin concentration, withless inhibition at lower concentrations (Fig. 7).NE-cleaved PAR2 Does Not Recruit �-Arrestin and Is

Retained on the Cell Surface—PAR2 activation by trypsin is fol-lowed by rapid receptor phosphorylation, recruitment of �-ar-restin to the receptor, and subsequent internalization andtargeting of the activated PAR2 either to lysosomes for degra-dation or to an intracellular scaffold for further signaling via

MAPK (12, 14, 25). A lack of �-arrestin recruitment would beexpected to result in a different signaling response and differ-ences in receptor trafficking compared with trypsin-activatedPAR2. In order to determine if NE-cleaved PAR2 interacts with�-arrestin, we transfected cells expressing YFP-tagged PAR2along with the Rluc-�-arr1 and employed an energy transfer(BRET) assay to detect receptor �-arrestin-1 interactions afterPAR2 activation. Trypsin and PAR2-activating peptide (2f-LIGRLO-NH2) stimulation of PAR2-expressing cells resulted ina substantial increase in the BRET ratio, indicating a recruit-ment of �-arrestin-1 to the activated receptor (Fig. 8A). How-ever, following activation of PAR2 with NE, we failed to observeany increase in the BRET ratio (Fig. 8A). Similar data were

FIGURE 5. NE disarms calcium signaling and activated p44/42 MAPK in HPT cells. HPT cells were incubated with the Fluo-4 no wash calcium indicator for30 min, and trypsin-stimulated calcium signaling was monitored in cells that were preincubated or not with 3 units/ml NE for 20 min. A, top, representative traceshowing trypsin signaling in HPT cells. Bottom, representative trace showing the lack of NE-stimulated calcium signaling in HPT cells and the subsequentattenuation of trypsin triggered calcium signaling in these cells (// indicates 20 min). The PAR2-AP SLIGRL-NH2 is still able to stimulate calcium signaling in cellsthat are disarmed by NE (mean � S.E. (error bars), n � 3; *, significant decrease in trypsin-stimulated calcium signal in HPT cells that were exposed to NEcompared with cells that were not preincubated with NE; p � 0.05). B, histogram showing trypsin-stimulated calcium signaling in HPT cells that werepreincubated or not with 3 units/ml NE as a percentage of calcium stimulated by the calcium ionophore A23187 (mean � S.E., n � 3). *, significant increase inp44/42 MAPK signal compared with no treatment (NT) (p � 0.05). C, representative image showing activation of p44/42 MAPK by SLIGRL-NH2, trypsin, and NEin HPT cells. D, histogram showing densitometry analysis of multiple scanned Western blot images to quantify the percentage increase over base line of p44/42activation by SLIGRL-NH2, trypsin, and NE in HPT cells (mean � S.E., n � 3). *, significant increase in P44/42 MAPK signal compared with the NT (p � 0.05).

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obtained when monitoring recruitment of �-arrestin-2 (datanot shown). The lack of �-arrestin recruitment to NE-activatedPAR2 correlated with a lack of internalization of the receptor incells that had been activated with NE (Fig. 9). As seen for NE,neither CG nor PR3 caused receptor internalization (Fig. 9,lower panels). In contrast, activation of cells with either trypsinor SLIGRL-NH2 resulted in loss of cell surface receptor expres-sion and increases in intracellular PAR2-YFP levels in endo-some-like structures (Fig. 9, upper panels).To determine if NE-stimulated p44/42MAPK activationwas

independent of �-arrestins, we further examined p44/42MAPK phosphorylation in embryonic fibroblasts from wildtype (MEFwt) and �-arrestin-1/2-null (MEF�arrDKO) mice.Trypsin, 2f-LIGRLO-NH2, and NE all promoted increasedphosphorylation of p44/42 MAPK (14-, 5-, and 6-fold overuntreated, respectively) inMEFwt. However, inMEF�arrDKO,whereas trypsin- and 2f-LIGRLO-NH2-stimulated p44/42MAPK phosphorylation was reduced by 90 and 80%, relativeto MEFwt, NE-stimulated p44/42 MAPK activation inMEF�arrDKO was not significantly different from that

observed in MEFwt (Fig. 8, B and C). Thus, NE-stimulatedPAR2 signaling to MAPK, in contrast with trypsin and 2f-LIGRLO-NH2, appears to be independent of �-arrestins.

DISCUSSION

The discovery that serine proteinases can signal to cells bycleaving and activating PARs via a unique tethered ligandmechanism established a novel paradigm for inflammatory cellregulation (26, 27). Our newwork described here demonstratesfor the first time that neutrophil elastase, an endogenouslyexpressed proteinase, can act as a biased agonist at PAR2. In aninflamed tissue, increased neutrophil infiltration and release ofneutrophil serine proteinases will probably shift the balance ofPAR2 signaling toward this newly identified signaling mecha-nism and could have important implications for our under-standing of how these receptors regulate inflammation.Our newdata broaden considerably, well beyond the trypsin-

related serine proteinase family, the spectrum of proteinasesthat can cause tissue responses via the PARs, including the pos-sibility of PAR2 biased signaling (MAPK versus calcium), dem-onstrated here for elastase but not for the other two neutrophilproteinases. Previous work demonstrating the disarming ofPAR2 by neutrophil and Pseudomonas elastases (6, 28) consid-ered only the silencing of the calcium signal and did not recog-

FIGURE 6. NE-stimulated p44/42 MAPK activation is Rho-kinase-depen-dent. PAR2-expressing KNRK cells were serum-starved and stimulated withNE for 10 min in the presence or absence of Rho-kinase inhibitors Y27362 andH1152. A, representative image showing activation of p44/42 MAPK by NE inPAR2-KNRK cells and the attenuation of NE-stimulated MAPK signal by Rho-kinase inhibitors Y27362 and H1152. B, histogram showing densitometryanalysis of multiple scanned Western blot images to quantify the percentageincrease over base line of PAR2-KNRK p44/42 activation by NE in the presenceor absence of Rho-kinase inhibitors. Increases in p44/42 MAPK (p-p44/42)phosphorylation were normalized to total p44/42 MAPK (t-p44/42) signal inthe same samples and expressed as a percentage increase over base-linevalues (mean � S.E. (error bars), n � 3). *, significant decrease in signal com-pared with the NE (3 units/ml) samples (p � 0.05). NT, no treatment.

FIGURE 7. Elafin inhibition of NE abrogates activation of PAR2-dependentp44/42 MAPK activation. PAR2-expressing KNRK cells were serum-starvedand stimulated with NE that had been preincubated with different concen-trations of elafin for 10 min. A, representative image showing activation ofp44/42 MAPK by NE in PAR2-KNRK cells and the attenuation of NE-stimulatedMAPK signal by the NE inhibitor elafin. B, histogram showing densitometryanalysis of multiple scanned Western blot images to quantify the percentageincrease over base line of PAR2-KNRK p44/42 activation by NE in the presenceor absence of elafin. Increases in p44/42 MAPK phosphorylation (p-p44/42)were normalized to total p44/42 MAPK (t-p44/42) signal in the same samplesand expressed as a percentage increase over base-line values (mean � S.E.(error bars), n � 3; *, significant decrease in signal compared with the NE (3units/ml) samples; p � 0.05).

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nize the potential impact on MAPK activation that our newobservations reveal. Interestingly, the mechanism of MAPKactivation also appears to be distinct from the trypsin-triggeredresponse. We observe that the elastase-activated receptor isimpaired in its ability to internalize and fails to interact with�-arrestin. �-Arrestin interaction is known to be important forPAR2 internalization (29). Further, �-arrestin scaffolds theinternalized receptor to Raf-1 and activated ERK-MAPK andensures cytosolic retention of the activated ERK (30), whereasERK-MAPK activation by PAR2 receptors that are unable to

interact with �-arrestin results in the nuclear translocation ofactivated ERK. These differences in localization of signalingcomplexes would suggest that the MAPK signaling pathwaytriggered byNE,which is independent of�-arrestin interaction,would result in ERK activation of nuclear transcription,whereas trypsin-activated PAR2 ERK-MAPK signaling wouldtarget non-nuclear substrates. This possible difference inMAPK signaling will probably impact on the inflammatoryresponse that PAR2 directs. The inhibition of the NE-triggeredMAPK signal by inhibitors of Rho-associated kinase indicatethat this response is occurring through the coupling of thereceptor to the G�12/13. The ability of PAR2 to engage G�12/13has been reported in a number of other cells (31, 32), but theimplications of selectively activating this arm of PAR2 signalingby NE remain to be fully understood.Although a number of studies have described the ability of

neutrophil-derived enzymes to inactivate PAR2-dependent cal-

FIGURE 8. NE-activated PAR2 receptors do not recruit �-arrestin-1 andactivate p44/42 MAPK independently of �-arrestin interaction. A, HEK-293 cells were transiently transfected with 1 �g of the PAR2-YFP constructalong with 0.1 �g of the Rluc-�-arr1 construct. Agonist-stimulated interactionbetween the PAR2 and �-arrestin-1 was detected over 60 min by measuringthe BRET ratio following the addition of 5 �M coelenterazine. NE (3 units/ml)did not stimulate recruitment of �-arrestin-1 to PAR2, whereas trypsin (10 nM)and the PAR2 agonist peptide 2f-LIGRLO-NH2 (10 �M) stimulated �-arrestinrecruitment to PAR2. B, representative Western blot showing p44/42 MAPKactivation in MEFwt and MEF�arrDKO stimulated with trypsin, 2f-LIGRLO-NH2, or NE. C, quantification of -fold change in phospho-p44/42 MAPK(p-P44/42 MAPK) over base line, where base line is defined as phospho-p44/42MAPK levels in untreated MEFwt (mean � S.E. (error bars), n � 3; *, significantincrease in phospho-p44/42 MAPK levels over nontreated (NT) samples (p �0.05); #, significant difference in phospho-p44/42 MAPK levels betweenMEFwt and MEF-�arrDKO (p � 0.05)).

FIGURE 9. NE activated PAR2 receptors are retained on the cell surface.A, PAR2-YFP-transfected HEK cells were plated on a glass bottom Petri dish,and agonist-stimulated internalization of the receptor was monitored by con-focal microscopy. NE-, CG-, and PR3-activated PAR2 receptors do not internal-ize and are retained on the cell surface, whereas trypsin- and SLIGRL-NH2-stimulated PAR2 receptors internalize and localize to endosome-likestructures. B, quantitative morphometric analysis of receptor internalizationdone as outlined under “Experimental Procedures” for either untreated cellsor cells treated with trypsin, SLIGRL-NH2, NE, PR3, or CG. Internalization ofreceptors, measured in three independently conducted experiments, isexpressed as the average number of internalized fluorescent speckles percell � S.E. (error bars).

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cium signaling (6, 7), few studies have described the ability ofthese enzymes to modulate cellular responses through thisreceptor. A recent study examining the regulation of epithelialbarrier permeability has shown that NE and PR3 activation ofPAR2 (and PAR1) on the basolateral aspect of colonic epithelialcells increases barrier permeability (33). The authors proposedthat this effect was amechanism for allowing increased neutro-phil transepithelial migration. More recently, in an in vivomouse model of colitis, it was shown that inhibition of NE byelafin significantly suppresses inflammatory mediators andstrengthens the intestinal epithelial barrier functions in colonictissues from mice as well as in intestinal epithelial cells (34).Given that PAR2 is believed to be a key player in the pathogen-esis of colitis (35, 36), it will be interesting to evaluate furtherthe signaling responses and trafficking of the receptor in thecolonic epithelial cells to assess the possible relationship to datain this report. Similarly our data in kidney proximal tubularcells, which express an abundance of PAR2, demonstrate thatneutrophil infiltration, which is a hallmark of renal inflamma-tion, will shift the balance of signaling via this receptor. ThisNE-mediated impact on tubular cell signaling in the kidneymaytrigger an epithelial to mesenchymal transition so as to play arole in renal fibrosis.The cleavage of the PAR2 N terminus downstream of the

TL-revealing site by the neutrophil proteinases can also becompared with our preliminary evaluation of their cleavage ofthe human PAR1-, PAR2-, and PAR4-derived sequences span-ning the TL domains (supplemental Table 1). NE and PR3 wereable to cleave the TL-spanning peptide of PAR1 at sites thatwould prevent subsequent activation by thrombin, and thusthis novel signaling mechanism that we have uncovered forPAR2 may reflect a general mechanism for PAR regulation byneutrophil proteinases. There is some evidence to suggest thatPAR1 can exhibit biased agonism. For instance, elastase, whichwe show cannot unmask the PAR1 TL sequence in vitro, isnonetheless able to induce apoptosis in human lung epithelialcells via a process that appears to be PAR1-mediated (37, 38).Further, platelet PAR1 was recently reported to be cleaved bythe metalloproteinase MMP-1 to reveal a “non-canonical”receptor-activating tethered ligand that is distinct from thatrevealed by thrombin (39). The MMP-1-dependent activationof PAR1 was shown to bias signaling toward the Rho-GTP andMAPK pathways. In addition, activated protein C has beenshown to cause a PAR1-mediated increase in endothelial barrierintegrity, whereas thrombin activation of PAR1decreases endo-thelial barrier function (40, 41). Functional selectivity of thecoupling of PAR1 to different G-protein-mediated responses,with differences in coupling between the receptor activation bythrombin and agonist peptide activation of the receptor, hasalso been reported (42). Whether neutrophil proteinase-medi-ated disarming of either PAR1 or PAR4 triggers biased signalingby these receptors, as we describe here for PAR2, is currentlyunknown. Nonetheless, the influx of inflammatory neutrophilsin response to injury or infection, via the actions of elastase,proteinase-3, and cathepsin-G acting on the PARs, couldin principle stimulate a complex set of responses by activatingPAR4 (cathepsin-G) (7, 43), disarming PAR1 and PAR2 (cathe-psin-G and proteinase-3) (7, 23), and selectively activating a

unique arm of the PAR2MAPK signaling pathway (elastase). Insummary, we identify in neutrophil elastase an endogenouslyexpressed biased enzyme agonist for PAR2. We also proposethat proteinase-triggered biased signaling via PAR2, shownhere for the first time for neutrophil elastase, may underpinthe distinct responses stimulated by PAR2 in inflammatorysettings.

Acknowledgments—We thank the live cell imaging core and themolecular instrumentation core laboratories of the Snyder Institute ofInfection, Immunity, and Inflammation at the University of Calgary.We thank Dr. Gerald Zamponi for access to the Mithras LB940 platereader for performing the BRET experiments and Dr. Ed Conway forcritically reading themanuscript.We also thankDr.WenjieWang forhelp with isolating human proximal tubular cells.

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Lau, Daniel A. Muruve, Kathryn A. DeFea, Michel Bouvier and Morley D. HollenbergRithwik Ramachandran, Koichiro Mihara, Hyunjae Chung, Bernard Renaux, Chang S.

)2(PARNeutrophil Elastase Acts as a Biased Agonist for Proteinase-activated Receptor-2

doi: 10.1074/jbc.M110.201988 originally published online May 16, 20112011, 286:24638-24648.J. Biol. Chem. 

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