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Originally published In Press as doi:10.1074/jbc.M310836200 on February 24, 2004

J. Biol. Chem., Vol. 279, Issue 18, 18592-18599, April 30, 2004
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Regulated Shedding of PAR1 N-terminal Exodomain from Endothelial Cells*

Matthew J. Ludeman{ddagger}, Yao Wu Zheng{ddagger}, Kenji Ishii§, and Shaun R. Coughlin{ddagger}||

From the {ddagger}Cardiovascular Research Institute and the Departments of Medicine and Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143-0130 and the §Department of Geriatric Medicine, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan

Received for publication, October 1, 2003 , and in revised form, February 24, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
G protein-coupled receptors can trigger metalloproteinase-dependent shedding of proteins from the cell surface. We now report that G protein-coupled receptors can themselves undergo regulated metalloproteinase-dependent shedding. The N-terminal exodomain of protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin, displayed regulated shedding in endothelial cells, which normally express this receptor. Cleavage occurred at a site predicted to render the receptor unresponsive to thrombin. A chimeric protein in which the N-terminal exodomain of PAR1 was fused to an unrelated transmembrane segment was shed as efficiently as PAR1, shedding of both proteins was stimulated by phorbol ester and by a PAR1 agonist. TNF{alpha} protease inhibitor-2 (TAPI-2), phenanthroline, and tissue inhibitor of metalloproteinase-3 (TIMP-3) but not TIMP-1 or -2 inhibited such shedding. These and other data suggest that the information that specifies PAR1 shedding resides within its N-terminal exodomain rather than its heptahelical segment, that activation of protein kinase C or of PAR1 itself can stimulate PAR1 shedding in trans, and that ADAM17/TACE or a metalloproteinase with similar properties mediates PAR1 shedding. Regulated shedding reduced the amount of cell surface PAR1 available for productive cleavage by thrombin by half or more, but thus far we have been unable to demonstrate an effect of PAR1 shedding on cellular responsiveness to thrombin. Nonetheless, regulated shedding of G protein-coupled receptors represents a new mechanism by which signaling by this important class of receptors might be modulated.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
G protein-coupled receptors (GPCRs)1 including protease-activated receptor-1 (PAR1), a G protein-coupled receptor that mediates thrombin signaling, can stimulate cleavage and "shedding" of the extracellular domains of a number of integral membrane proteins. Several such cleavage events are mediated by membrane-bound metalloproteinases of the ADAMs (a disintegrin and metalloprotease) family (1). For example, ADAM17 (TNF{alpha}-converting enzyme or TACE) mediates cleavage and release of HB-EGF from cells, and HB-EGF shedding can be activated by PAR1 and other GPCRs (2, 3). Regulated shedding of cell surface proteins may be important for embryonic development (4, 5), leukocyte-vessel wall interaction (4, 6), and other functions that involve release of the exodomains of growth factors and cytokines, adhesion molecules, receptors, and other integral membrane proteins from the cell surface (7–13).

PAR1 is the main thrombin receptor in human platelets and probably plays an important role in hemostasis and thrombosis (14, 15). PAR1 is also the main thrombin receptor on vascular endothelial cells, and activation of endothelial PAR1 by thrombin and related proteases triggers a host of signaling responses that may participate in inflammation and contribute to embryonic development (16–19).

In the course of beginning to probe the signaling pathways by which PAR1 controls the shedding of other cell surface proteins, we noted that a chimeric protein in which the N-terminal exodomain of PAR1 was used to tether alkaline phosphatase (AP) to the cell membrane displayed regulated shedding. The possibility that the N-terminal exodomain of PAR1 might be subject to shedding in the context of the receptor itself was of interest given the mechanism by which PAR1 is normally activated (Fig. 1A). Thrombin recognizes and cleaves the N-terminal exodomain of PAR1 between Arg41 and Ser42. This cleavage event generates a new receptor N terminus that serves as a tethered peptide ligand that docks with the receptor heptahelical segment to cause transmembrane signaling and G protein activation (14). In accord with this model, deletion of the PAR1 N-terminal exodomain in a manner that removes the tethered ligand by either mutation or proteolysis renders PAR1 insensitive to thrombin (20–24). We therefore investigated the possibility that regulated shedding of the N-terminal exodomain of PAR1 by cleavage C-terminal to the tethered ligand might occur and its possible significance for regulation of PAR1 function.



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  		FIG. 1.
Mechanism of PAR1 activation and assay for PAR1 shedding. A, mechanism of activation. Thrombin-mediated cleavage and activation of native PAR1. Thrombin (large sphere) binds to (1) and cleaves PAR1 to unmask a tethered ligand (diamond) (2), which then binds to and activates the receptor (3). Note that removal of the PAR1 N-terminal exodomain would render the receptor unresponsive to thrombin. B, membrane-associated metalloproteinase (large oval) cleaves N-terminal exodomain tagged at its N terminus with alkaline phosphatase. Such cleavage releases alkaline phosphatase activity into the culture medium. Constructs were made to allow assay of cleavage or shedding of the PAR1 N-terminal exodomain in the context of the native receptor (AP-PAR1) and tethered to an unrelated transmembrane domain (AP-ATE-CD8).

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Phorbol 12-myristate, 13-acetate (PMA), 1,10-phenanthroline, and other reagents were from Sigma-Aldrich unless noted. Human {alpha}-thrombin was from Enzyme Research Laboratories (South Bend, IN). Peptide SFLLRN was synthesized as a C-terminal amide and purified by high pressure liquid chromatography (ANASPEC, San Jose, CA). TAPI-2 was from Peptides International (Louisville, KY). Recombinant human TIMPs 1, 2, and 3 were from R&D Systems (Minneapolis, MN). Ecotin, E-64, and pepstatin A were generous gifts from Dr. Charles Craik (University of California, San Francisco). Cells, cell culture media, and supplements were from University of California, San Francisco Cell Culture Facility unless otherwise specified.

Antibodies—Rabbit antiserum 1809, "anti-hirudin-like sequence antibody," was raised against the peptide antigen YEPFWDEEKNESGLTEY (25). ATAP2, a mouse monoclonal antibody to PAR1 N-terminal exodomain (26), was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-alkaline phosphatase monoclonal antibody was from Sigma. Horseradish peroxidase-conjugated goat anti-rabbit and goat antimouse antibodies were from Bio-Rad, except in assays for detection of soluble native PAR1 N-terminal exodomain by ELISA, in which horseradish peroxidase-conjugated goat anti-rabbit antibodies were from Zymed Laboratories (South San Francisco, CA).

Plasmid cDNAs—AP-PAR1 encoded secreted human placental alkaline phosphatase (SEAP) fused to human PAR1 at Ala36 with an intervening linker region containing the FLAG epitope (DYKDDDD). The amino acid sequence of the fusion region reads:... AAHPGLQDYKDDDDVD-ATLDPR{downarrow}SFLLR... where the alkaline phosphatase sequence is underlined, (-) marks the linker junction to native PAR1 sequence shown in bold, and ({downarrow}) indicates the thrombin cleavage site. Additionally, sequence 5' of the SEAP Ile18 codon had been replaced with a secretory sequence derived from mast cell growth factor that included a Myc epitope tag. AP-PAR1 cDNA was inserted into the mammalian expression vector, pcDNA3 (Invitrogen), at HindIII/NotI cloning sites.

AP-ATE-CD8 was derived from a previously described PAR1 mutant, ATE-CD8 (27). Briefly, ATE-CD8 encodes the FLAG epitope-tagged human PAR1 amino terminal exodomain, up to and including residue Asp91, fused to the extracellular aspect of the CD8 single transmembrane domain at CD8 residue Ile162. To make AP-ATE-CD8, a PCR product beginning at the ATE-CD8 5'-FLAG epitope codon and ending at the stop codon was generated with flanking PvuII and XbaI restriction sites, respectively. The PvuII-XbaI fragment was ligated into the eukaryotic expression vector, pCMV/SEAP (Tropix, Bedford, MA) at the HpaI and XbaI restriction sites, resulting in a fusion product containing the entire SEAP coding sequence (excluding the stop codon) joined by a FLAG epitope-containing linker sequence to the PAR1 Ala36 of ATE-CD8. The predicted amino acid sequence of the product is:... AAHPGSDYKDDDDVD-ATLDPR{downarrow}SFLLRNPNDKYEPFWEDEEKNESGLTEYRLVSINKSSPLQKQLPAFISED-IYIWA... Labeling is as above for AP-PAR1; the CD8 sequence is italicized.

AP-PAR4 was derived from a FLAG epitope-tagged human PAR4 construct in a manner directly analogous to AP-ATE-CD8. A PCR product beginning at the 5' FLAG epitope codon and ending at the stop codon was generated with flanking PvuII and XbaI restriction sites, and the PvuII-XbaI fragment ligated into pCMV/SEAP as above, resulting in a fusion product containing the SEAP coding sequence (excluding the stop codon) joined by a FLAG epitope-containing linker sequence to PAR4 Val25. The predicted amino acid sequence of the junction is:... AAHPGSDYKDDDDVDAL-VYDESGSTGGGDDSTPSILPAPR{downarrow}G-YPGQV...; labeling is as above for AP-PAR1. The sequence of the junctions regions for AP-PAR1, AP-ATE-CD8, and AP-PAR4 were confirmed by dideoxy sequencing.

Cells, Cell Culture, and Shedding Assays—All cells were cultured and incubations conducted at 37 °C in a 5% CO2, humidified atmosphere. Human umbilical vein endothelial cells (HUVECs; Clonetics, San Diego, CA), were maintained in Clonetics EGM-2 complete growth media and cultured on gelatin-coated Falcon cell culture 24-well plates (BD Biosciences, Franklin Lakes, NJ). Chinese hamster ovary CHO-M1 mutant cells were a generous gift from Dr. Joan Massague (Memorial Sloan-Kettering Hospital, New York). The ATE7/CHO cell line stably expressing AP-ATE-CD8 was generated by transfecting wild-type CHO cells with AP-ATE-CD8 cDNA, selecting G418-resistant clones (neo is contained in the AP-ATE-CD8 vector), exposing each to PMA, then screening for the presence of alkaline phosphatase activity in the culture media.

CHO-M1 cells, ATE7/CHO cells, and wild-type CHO cells were maintained in F-12 Ham's nutrient mix medium supplemented with 10% fetal bovine serum (heat-inactivated), penicillin (100 units/ml), streptomycin (100 µg/ml), and fungizone (0.25 µg/ml), and were plated on Falcon Primaria 24-well or 96-well plates. For CHO-M1 mutant and wild-type CHO cell assays, cells were transiently transfected in 24-well plates with LipofectAMINE-PLUS (Invitrogen) according to the manufacturer's instructions 2 days before the assay. To examine shedding, cells were incubated in serum-free medium (for CHO cells, Ham's F-12 with 15 mM HEPES and 0.1% bovine serum albumin) overnight. Cells were then washed and further incubated in serum-free medium in the presence or absence of TAPI-2 or other protease inhibitors for 15–20 min prior to the addition of agonist.

For endothelial cell assays in which AP-tagged proteins were used, passage 5 or 6 HUVECs at 80–85% confluence in 24-well plates were transiently transfected with AP-PAR1 or AP-ATE-CD8 in antibiotic-free EGM-2 complete growth medium using the polyamine transfection reagent, TransIT-LT1 (Mirus Corp., Madison, WI), according to the manufacturer's instructions. On the following day, the cells were washed and incubated in serum-free medium (for HUVECs, Clonetics basal EBM-2 media with 15 mM HEPES and 0.1% bovine serum albumin) for 5 h, washed again, and preincubated with inhibitor or control medium alone for 15–20 min prior to the addition of agonist. In all cases, cells were incubated for an additional 45 or 60 min, as indicated, after which conditioned medium was collected, spun at 14,200 x g for 2 min at 4 °C, then assayed for alkaline phosphatase activity (see below). To determine the amount of intact "unshed" receptor remaining on the cell surface and available for activation by thrombin after agonist-stimulated shedding, cells were washed three times and then exposed to 10 nM thrombin for an additional 20 min, at which time supernatant was collected and shed product was again measured.

To assay shedding of native PAR1, passage 5 or 6 HUVECs were grown to confluence on gelatin-coated 60-mm dishes. The cells were then washed and incubated in serum-free medium for 5 h as described above for transfected HUVECs. Cells were then washed again and incubated in the presence or absence of TAPI-2 for 15–20 min. Agonist or medium alone was then added, and the cells incubated for an additional 60 min, at which time 1.8 ml (of a total 2.2 ml) of conditioned medium was collected and centrifuged at 14,200 x g for 2 min at 4 °C. Approximately 1.4 ml of the cleared conditioned medium was concentrated to a volume of ~150 µl using Microcon Centrifugal filter units with a 3,000-dalton cut-off (Millipore) and assayed for the presence of soluble native PAR1 N-terminal exodomain (see below).

Measurement of Shed AP-tagged Product—The amount of alkaline phosphatase shed from the cell surface was determining by measuring alkaline phosphatase activity present in the culture medium of treated cells expressing the AP-tagged proteins (28). AP activity was quantified using the Phospha-LightTM chemiluminescent reporter assay for secreted alkaline phosphatase kit (Tropix) and a Tropix TR717 microplate luminometer according to the manufacturer's instructions. After centrifugation, 50 µl of conditioned medium was added to 150 µl of dilution buffer (0.l M diethanolamine, 1 mM MgCl2) and heated to 65 °C for 30 min (to inactivate endogenous alkaline phosphatase). After briefly cooling the heat-treated sample, 50 µl was added to 50 µl of assay buffer (proprietary mixture of phosphatase inhibitors in 20 mM Tris, pH 9.8, 1 mM MgCl2) and allowed to equilibrate at room temperature for 5 min, at which time 50 µl of reaction buffer, containing 1.25 mM CPSD (chemiluminescent substrate, disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)phenyl phosphate), was added, and the sample was analyzed for chemiluminescence.

Subpicogram quantities of recombinant AP could be detected by this assay, and the relationship between chemiluminescence and protein was linear from 0.1 pg to at least 10 ng. Activity levels in the experiments presented here generally ranged from 5 pg to 1 ng. Background luminescence from untransfected cells was equivalent to less than 1 pg.

Cell Surface Antibody Binding Assay—Binding of anti-hirudin-like sequence and anti-AP antibodies to cells was measured as previously described (25) and was dependent upon transfection of cells with AP-ATE-CD8. Wild-type CHO cells were transiently transfected with AP-ATE-CD8 cDNA using LipofectAMINE-PLUS as above 2 days before the assay. Cells were then incubated overnight in serum-free medium, washed twice, and then preincubated for 15–20 min in the presence or absence of TAPI-2 prior to the addition of agonist. After an additional 45 min, cells were washed once in phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. After fixation, cells were washed twice in serum-free medium, incubated with either anti-AP mAb (1:1,000) or anti-hirudin-like sequence (1809) antiserum (1:200) for 1 h, washed three times, incubated with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibody (1:1000) for 30 min, and then washed three times (PBS was used for the final rinse). Immediately following the final rinse, the cells were incubated with the horseradish peroxidase substrate 1-Step ABTS (2, 2'-azino-bis-3-ethylbenz-thiazoline-6-sulfonic acid) (Pierce). After 10 min, an aliquot was removed, and the optical density was read at 405 nm using a Molecular Devices microplate spectrophotometer (Molecular Devices, Menlo Park, CA).

ELISA for Shed PAR1 N-terminal Exodomain—To detect shed native PAR1 N-terminal exodomain in conditioned medium, a two-antibody capture and detection method was used. The mouse monoclonal antibody ATAP2, which was raised to a peptide SFLLRNPNDKYEPF corresponding to the 14 residues that constitute the new PAR1 N terminus after thrombin cleavage (26) was adsorbed to 96-well MaxiSorp plates (Nalge Nunc International, Naperville, IL) by incubating plates with antibody (5 µg/ml in PBS) at 4 °C for 48 h. Wells were washed three times and blocked with M199 medium containing 20 mM HEPES and 0.5% bovine serum albumin for 1–3 h at room temperature, then washed once more with blocking solution, and incubated with 150 µl of concentrated conditioned medium (see above) overnight at 4 °C. The following day, plates were washed three times with PBS supplemented with 0.2 M NaCl and 0.05% Tween 20, incubated with anti-hirudin-like sequence (1809) antiserum (1:200) for 1 h at room temperature, washed three times, incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (0.6 µg/ml) for 1 h, and then washed three times (PBS with 0.05% Tween 20 was used as diluent for both antibodies). Immediately following the final wash, 1-step ABTS was added to the wells, and optical density was read at 405 nm after ~30 min.

A purified, bacterially expressed recombinant soluble form of the PAR1 N-terminal exodomain in fresh medium was used as a standard to assess the selectivity and sensitivity of this system. The peptide, dubbed ATE-H6, and previously described (21), contained the antigenic regions for both the capture (ATAP2) and detection (anti-hirudin-like sequence) antibodies. The ELISA signal was approximately linear between peptide concentrations of ~3 and 60 pg/ml, and little signal was obtained in the absence of either the capture antibody or the detection antibody (not shown).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To study GPCR-regulated shedding, we generated a number of vectors to direct expression of chimeric proteins in which secreted AP was fused to the extracellular N terminus of a transmembrane protein. Among these was AP-ATE-CD8, a chimeric protein in which secreted alkaline phosphatase was fused to the N-terminal exodomain of PAR1, which was in turn joined to the transmembrane domain of CD8 (Fig. 1). The resulting protein tethered alkaline phosphatase to the cell surface via the PAR1 N-terminal exodomain such that proteolytic cleavage of that domain by thrombin released alkaline phosphatase into the culture medium. AP-ATE-CD8 was intended for use in developing an assay for regulated shedding with thrombin cleavage acting as a positive control. As expected, treatment of CHO cells transfected with AP-ATE-CD8 with 10 nM thrombin led to a rapid increase in soluble alkaline phosphatase activity in the culture medium, with over 90% of cell surface alkaline phosphatase being released within 10 min. Virtually no alkaline phosphatase activity was released from untransfected cells. Surprisingly, we also noted that exposure of AP-ATE-CD8-expressing CHO cells to the protein kinase C activator PMA triggered the appearance of alkaline phosphatase activity in the medium (Fig. 2). The increase in soluble alkaline phosphatase activity was linear for ~60 min after addition of PMA, and peak activity at this time ranged from 50 to 100% of that released by thrombin. Concentrations of the thrombin inhibitor hirudin that completely blocked thrombin-induced shedding had no effect on PMA-induced shedding (not shown), suggesting that AP-ATE-CD8 was being cleaved by a protease other than thrombin in this system. PMA is known to trigger shedding of the exodomains of integral membrane proteins by a metalloproteinase-dependent mechanism. Indeed, the metalloproteinase inhibitors TAPI-2, 1,10 phenanthroline (1,10-PA), and TIMP-3 blocked PMA-induced shedding and reduced unstimulated shedding of AP-ATE-CD8 (Fig. 2). At the concentrations used in these experiments, neither TAPI-2 nor TIMP-3 had any effect on thrombin-induced shedding (Fig. 2 and data not shown). The serine, cysteine, and aspartyl protease inhibitors ecotin, E-64, and pepstatin A had no effect on PMA-induced shedding (Fig. 2). Taken together, these data suggested that AP-ATE-CD8 was undergoing regulated metal-loproteinase-dependent shedding.



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  		FIG. 2.
Sensitivity of PAR1 N-terminal exodomain shedding to protease inhibitors. CHO cells stably expressing AP-ATE-CD8 (ATE7/CHO) were treated with PMA (1 µM) for 1 h in the presence or absence of the following inhibitors: A, ecotin (10 µM); E-64 (50 µM); pepstatin A (1 µM); and TAPI (50 µM); and B, TIMP-1 (10 µg/ml); TIMP-2 (10 µg/ml); TIMP-3 (10 µg/ml); 1,10-phenanthroline (5 mM), and TAPI-2 (50 µM). Cells were also incubated with thrombin (10 nM)to determine total releasable alkaline phosphatase activity from AP-ATE-CD8-expressing cells. Results expressed as fraction of total thrombin-releasable alkaline phosphatase activity (mean ± S.E.) from two replicate experiments are shown. Individual points were done in triplicate in each experiment.

 
In contrast to TIMP-3, TIMP-1, and TIMP-2 did not block PMA-induced shedding in this system. This pattern of inhibitor specificity is consistent with the possibility that the metalloproteinase responsible for AP-ATE-CD8 shedding is TACE (30). To further test this possibility, we examined shedding in CHO-M1 cells, a CHO variant in which TACE biogenesis is defective (31). Both AP-ATE-CD8 and AP-PAR1, a construct in which alkaline phosphatase was fused to the N-terminal exodomain of human PAR1 six codons N-terminal to the thrombin cleavage site (Fig. 1), were examined. Basal shedding of AP-ATE-CD8 and AP-PAR1 in CHO-M1 cells was similar to that seen in wild-type CHO cells, but, in contrast to wild-type CHO cells, no PMA-induced shedding was detected in the CHO-M1 line (Fig. 3 and data not shown). Thus the N-terminal exodomain of PAR1 can undergo PMA-regulated metalloproteinase-dependent shedding in the context of the full-length receptor just as it does in AP-ATE-CD8, and this may be mediated by TACE or another metalloproteinase with similar inhibitor specificity and similar requirements for proper biogenesis.



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  		FIG. 3.
PMA induced shedding of PAR1 in wild-type and mutant CHO cells (CHO-M1) defective in TACE biosynthesis. Cells transiently transfected with AP-PAR1 were treated with PMA (1 µM) for 45 min in the presence or absence of TAPI-2 (50 µM), and alkaline phosphatase activity in supernatant was measured and expressed as fold basal activity released from AP-PAR1-transfected wild-type cells. Results represent three replicate experiments. Individual points were done in triplicate in each experiment.

 
The site within PAR1 that is recognized and cleaved by thrombin as well as the tethered ligand that triggers transmembrane signaling reside in the sequence LDPR{downarrow} SFLLRNPNDKYEPFWEDEE in the N-terminal exodomain of the receptor. This sequence represents receptor amino acids 38–60; {downarrow} indicates location of the peptide bond cleaved by thrombin, and SFLLRN is the tethered ligand. To determine whether these functionally important sequences are removed from the receptor during shedding, we examined loss of binding sites for antibodies that recognize the C-terminal portion of this sequence (peptide antigen YEPFWEDEEKNESGLTEYC, the so-called hirudin-like sequence, see "Materials and Methods"). Loss of binding sites for an antibody to AP, which is at the extreme N terminus of the target protein, was determined in parallel. AP-ATE-CD8 was chosen as the target protein because, in contrast to the full-length receptor, it undergoes relatively little internalization in response to either PMA or thrombin activation. Exposure of AP-ATE-CD8-expressing CHO cells to PMA yielded a 70–80% decrease in both AP and hirudin-like sequence binding sites, and both decreases were inhibited by TAPI-2 (Fig. 4). By contrast, exposure to thrombin led to a virtually complete loss of the AP epitope, which is N-terminal to the thrombin cleavage site, but only an ~45% decrease in hirudin-like sequence binding sites. TAPI-2 had no effect on thrombin-triggered loss of the AP epitope but did partially inhibit thrombin-triggered loss of the hirudin-like sequence epitope. The parallel decrease in the AP and hirudin-like sequence epitopes trigged by PMA, and the parallel inhibition of this decrease by TAPI-2 is consistent with metalloproteinase-dependent cleavage of the PAR1 N-terminal exodomain at a site C-terminal to the hirudin-like sequence. By contrast, the greater loss of the AP epitope compared with the hirudin-like sequence epitope in response to thrombin and the insensitivity of this loss of the AP epitope to TAPI-2 is consistent with thrombin cleaving the PAR1 N-terminal exodomain at the known thrombin cleavage site between the AP and hirudin-like sequence epitopes. The partial decrease in surface hirudin-like sequence epitope in response to thrombin may be due to some shedding caused by activation of endogenous thrombin receptors in CHO cells (see below) together with some internalization and/or non-productive cleavage at other sites. These data suggest that metalloproteinase-dependent cleavage of the PAR1 N-terminal exodomain occurs between the receptor hirudin-like sequence and the plasma membrane. Because a receptor processed in this manner would lack a tethered ligand, it would be incapable of mediating signaling to thrombin.



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  		FIG. 4.
Loss of alkaline phosphatase versus hirudin-like sequence epitopes from the cell surface. CHO cells transiently transfected with AP-ATE-CD8 were treated with PMA (1 µM), or thrombin (10 nM), for 45 min in the presence or absence of TAPI-2 (50 µM), and binding of antibody to AP (A) or hirudin-like sequence (B) was determined. Results (mean ± S.E.) are expressed as fraction of binding to untreated cells and represent two replicate experiments. Individual points were done in triplicate in each experiment.

 
We next asked whether regulated shedding might be a general property of PAR N-terminal exodomains. A construct analogous to AP-PAR1 was made for the other thrombin receptor, PAR4, which is primarily expressed on platelets. Alkaline phosphatase activity was readily released when cells expressing the AP-PAR4 construct were exposed to thrombin. By contrast, PAR4 exhibited low basal and PMA-induced release of alkaline phosphatase activity compared with PAR1 (expressed as a fraction of the total alkaline phosphatase activity released by thrombin, PAR4 showed basal and PMA-induced shedding of ~4 and ~6%, respectively, versus ~12 and ~35% for PAR1) (Fig. 5). Given that the N-terminal exodomain of PAR1 showed efficient shedding when tethered to the transmembrane domain of CD8 or to PAR1 itself, these data suggest that the specific amino acid sequence and/or length of the PAR N-terminal exodomains specifies their susceptibility to regulated shedding.



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  		FIG. 5.
Substantial metalloproteinase-mediated shedding of PAR1 versus PAR4 N-terminal exodomains in CHO cells. CHO cells in which AP-PAR4 or AP-PAR1 were transiently expressed were treated with PMA (1 µM) for 1 h in the presence or absence of TAPI-2 (50 µM) and alkaline phosphatase activity in the conditioned medium was measured. Results (mean ± S.E.) are expressed as a fraction of alkaline phosphatase activity released by 10 nM thrombin in 1 h and represent two replicate experiments. Individual points were done in duplicate or triplicate.

 
The data described above raised the question of whether regulated shedding of PAR N-terminal exodomains might serve as a mechanism for modulating PAR signaling. Toward addressing this question, we first asked whether the N-terminal exodomain of PAR1 undergoes metalloproteinase-dependent PMA-induced shedding in HUVECs, a thrombin-responsive primary cell that normally expresses PAR1 (32, 33). Soluble alkaline phosphatase was measured in conditioned medium from AP-ATE-CD8- and AP-PAR1-transfected HUVECs exposed to the PAR1/PAR2 agonist SFLLRN, PMA, or thrombin for 1 h (Fig. 6A). SFLLRN and PMA both triggered substantial increases in soluble alkaline phosphatase activity in the HU-VEC-conditioned medium in a TAPI-2-sensitive manner. The ability of SFLLRN to trigger shedding of the AP-ATE-CD8 construct (Fig. 6A), presumably by activating endogenously expressed wild-type PAR1 on the cell surface, suggests that PAR and presumably other GPCR signaling can trigger PAR1 N-terminal exodomain shedding in trans.



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  		FIG. 6.
Induced shedding of PAR1 in HUVECs and its effect on the number of receptors available on the cell surface for subsequent cleavage by thrombin. A, HUVECs transiently transfected with AP-PAR1 and AP-ATE-CD8 expression vectors were treated with PMA (1 µM), PAR1 agonist (SFLLRN; 100 µM), or thrombin (10 nM) for 1 h in the presence or absence of TAPI-2 (50 µM), and alkaline phosphatase activity in the conditioned medium was measured. Results are expressed as a fraction of alkaline phosphatase activity released by thrombin (mean ± S.E.) and represent three replicate experiments; individual points were done in triplicate in each experiment. B, cells in A (or identically treated cells) were washed three times after the initial incubation with agonists, and then treated with thrombin (10 nM) for an additional 20 min. Conditioned medium was again collected and assayed for alkaline phosphatase activity. Results (mean ± S.E.) are expressed as a fraction of alkaline phosphatase activity releasable from cells that were not exposed to agonist during the initial incubation and represent three separate experiments; individual points were done in triplicate in each experiment. Background luminescence from untransfected cells was subtracted in each experiment.

 
To address the possibility that regulated shedding of the PAR1 N-terminal exodomain might be an artifact of overexpression of the transfected reporter proteins, we utilized an ELISA to detect soluble PAR1 N-terminal exodomain in medium conditioned by untransfected HUVECs (Fig. 7). Incubation of the cells with PMA caused a TAPI-sensitive increase in soluble PAR1 N-terminal exodomain antigen in the conditioned medium. The magnitude of the PMA-induced signal was similar to that obtained by adding ~20 pg/ml of recombinant PAR1 N-terminal exodomain to the conditioned medium; this is consistent with ~2000 receptors per cell undergoing shedding, a plausible order of magnitude. The antibody used to capture the soluble PAR1 N-terminal exodomain (ATAP2) recognizes an epitope C-terminal to the PAR1 thrombin cleavage site, and the antibody used to detect it recognizes the hirudin-like sequence in PAR1 (Fig. 7 and Refs. 25 and 26). Thus the shed fragment contains the receptor hirudin-like sequence, suggesting that shedding is indeed mediated by cleavage C-terminal to the latter. Taken together, these data suggest that the PAR1 N-terminal exodomain can undergo metalloproteinase-mediated regulated shedding in untransfected endothelial cells in a manner that might reduce the number of receptors available for subsequent productive cleavage by thrombin.



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  		FIG. 7.
Induced shedding of native PAR1 in untransfected HU-VECs. Untransfected HUVECs were treated with PMA (1 µM)for 1h in the presence or absence of TAPI-2 (50 µM). Soluble native PAR1 N-terminal exodomain in the conditioned medium (concentrated ~9 times) was measure by ELISA. Results (mean ± S.E.) are expressed as fold over the basal signal obtained in medium from untreated cells and represent two replicate experiments (n = 5 or 6). Similar results were obtained in a third study.

 
To test the foregoing prediction, cells from the experiment described in the legend to Fig. 6A were washed thoroughly after the 1-h incubation with agonists and then exposed to thrombin for an additional 20 min. Soluble alkaline phosphatase activity released into the conditioned medium by thrombin in this second incubation was measured (Fig. 6B). Exposure of AP-PAR1-expressing HUVECs to thrombin during the initial incubation yielded cells that had virtually no remaining thrombin-releasable alkaline phosphatase activity, and this depletion was insensitive to TAPI-2. By comparison, exposure of cells to SFLLRN or PMA caused a sizeable but incomplete loss (~60–70%) of PAR1 available for subsequent cleavage by thrombin. This decrease was also seen with the ATE-CD8 construct and was substantially inhibited by TAPI-2. Thus metalloproteinase-dependent shedding is capable of decreasing the amount of PAR1 available for productive cleavage and, presumably, activation by thrombin.

Because regulated shedding of the PAR1 N-terminal exodomain would render the receptor insensitive to thrombin, we examined signaling in response to thrombin in HUVECs pre-incubated with PMA. Endpoints were intracellular calcium mobilization and phosphoinositide hydrolysis. PMA pretreatment did result in decreased thrombin responsiveness as measured by intracellular calcium flux. However, this effect was not blocked by TAPI-2 and hence could not be ascribed to metalloproteinase-dependent shedding of PAR1 (data not shown). Moreover, when measured by phosphoinositide hydrolysis, thrombin signaling in HUVECs was not diminished by pretreatment with PMA (not shown). Thrombin responses in HU-VECs are PAR1-dependent (16, 33, 34); thus, at face value, the level of shedding achieved under the conditions employed in our studies is insufficient to attenuate cellular responses to thrombin, at least as measured by intracellular calcium flux and phosphoinositide hydrolysis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have demonstrated that the N-terminal exodomain of the G protein-coupled thrombin receptor PAR1 can be shed from the endothelial cell surface in a regulated manner. Our results suggest that TACE or a TACE-like protease mediates such shedding. TACE is constitutively expressed by endothelial cells and is known to mediate shedding in response to activation of GPCRs or phorbol esters (7, 35). Regulated shedding of VCAM-1 in endothelial cells was recently shown to be mediated by TACE (6). Endothelial cells can express another ADAM that is known to be activated by GPCR signaling, ADAM10/Kuz (36, 37). However, ADAM10 is inhibited by TIMP-1 (38), which had no effect on PMA-induced shedding of PAR1 in our system. Additionally, it was recently shown that biosynthesis of TACE, but not that of several other surface metalloproteinases including ADAM10, was defective in a mutant cell line, CHO-M2. CHO-M2 and the mutant cell line used in our study, CHO-M1, are of the same genetic complementation group (with respect to shedding of the TACE substrate, TGF-{alpha}) and share a lack of properly processed TACE and inability to shed numerous known TACE substrates (31). Attempts to more directly confirm the role of TACE in regulated PAR1 shedding using TACE-knockout cells (4) were not interpretable. No inducible PAR1 shedding was detected in a fibroblast-like cell line derived from TACE knockout mice, but inducible shedding was also not detected in these same cells reconstituted with heterologously expressed TACE or in second fibroblast cell line from a mouse in which the TACE locus had not been manipulated (data not shown) (knockout cells and TACE cDNA were generously provided by Dr. Roy Black of Immunex Corp; second cell line was from a PAR1 knockout mouse and was stably transfected with a tagged PAR1). This result raises the possibility that regulated shedding of PAR1 may exhibit at least some cell type specificity.

The physiological importance of metalloproteinase-mediated shedding of the PAR1 N-terminal exodomain remains to be determined. The observations shown in Fig. 7 suggest that such shedding of native PAR1 does occur in untransfected endothelial cells. Moreover, shedding of PAR1 appears to occur by cleavage carboxyl to the tethered ligand of the receptor and hence provides a potential mechanism for disabling the receptor. Co-expression of PAR2 and AP-PAR1 in CHO cells resulted in shedding of AP-PAR1 in response to the PAR2 agonist SLIGRL; SLIGRL also triggered shedding of AP-PAR1 via endogenous PAR2 in COS-7 cells (data not shown). The observations that shedding can be stimulated by an activator of protein kinase C (PMA) and by activation of PAR1 and other GPCRs raise a number of possibilities.

One might imagine that regulated shedding could provide a mechanism for homologous desensitization by which PAR1 activation would rapidly decrease cellular sensitivity to thrombin, or perhaps even a mechanism for shutting off PAR1 signaling by stimulating removal of the tethered ligand from activated receptors. Kinetic considerations suggest that these roles are unlikely. Physiologically reasonable concentrations of thrombin (1–10 nM) cleave and activate the majority of PAR1 molecules on a cell surface within a few minutes, consistent with the rapid tempo of many biologically important responses to thrombin. Signaling by individual PAR1 molecules is also terminated within minutes by a mechanism that depends upon receptor phosphorylation (29), and desensitization of cells to thrombin occurs over the same time frame (16, 25, 33). By contrast, induced shedding of the PAR1 N-terminal exodomain by either PMA or by PAR1/PAR2 activation took ~30 min to reach half-maximal levels, and at 1 h only 60–70% of receptor N-terminal exodomains were shed. Thus shedding of the PAR1 N-terminal exodomain appears to be too slow to contribute to homologous desensitization or shutoff of signaling. In accord with this, TAPI-2 did not alter the tempo of thrombin-induced increases in intracellular calcium in HUVECs, nor did it inhibit desensitization of this response or augment other responses such as phosphoinositide hydrolysis or p44/42 mitogen-activated protein kinase (extracellular signal-regulated protein kinase) activation (data not shown).

One might also imagine that induced shedding of the PAR1 N-terminal exodomain would provide a mechanism for heterologous desensitization of cellular responsiveness to thrombin. That is, thrombin responsiveness might be dampened by prior "disarming" of a fraction of PAR1 molecules by shedding. Induced shedding did decrease the level of PAR1 available for activation by thrombin by as much as 70% in HUVECs (Fig. 6). As noted in the results section, we were unable to detect a substantial effect of such shedding on signaling. This study used PMA to trigger shedding, and this agonist triggers a variety of signaling responses that partially overlap those triggered by PAR1. Thus exposure of cells to PMA might well augment or desensitize responses to subsequent PAR1 activation by mechanisms independent of shedding. At face value, however, our studies suggest that the level of PAR1 shedding achieved in cultured HUVECs is insufficient to have a major impact on cellular responsiveness to thrombin. It is certainly possible that under other conditions or in other cell types, a level of PAR1 shedding sufficient to decrease thrombin responsiveness might be achieved.

It is formally possible that shedding of the N-terminal exodomain of PAR1 from the cell surface might provide a mechanism for generating a soluble peptide with a signaling function, analogous to shedding of TNF{alpha} or HB-EGF. However, micromolar soluble SFLLRN is required to activate PAR1. Thus the concentration of soluble tethered ligand generated by such a mechanism would almost certainly be insufficient to activate PAR1. Any signaling function of the released sequence would likely be novel and PAR1-independent.

Finally, our results demonstrate that, in addition to regulating sheddase activity, G protein-coupled receptors can themselves be substrates for sheddases. The extent to which this phenomenon is generalized and whether cleavage of GPCRs by sheddases is a physiologically important modulator of signaling remains to be determined.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants HL44907, HL59202, HL65185, and HL65590. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: University of California, San Francisco, HSE-1300, 513 Parnassus Ave., San Francisco, CA 94143-0130. E-mail: coughlin{at}cvrimail.ucsf.edu.

1 The abbreviations used are: GPCR, G protein-coupled receptor; ADAM, a disintegrin and metalloproteinase; PAR, protease-activated receptor; ATE, amino-terminal exodomain; AP, alkaline phosphatase; TNF{alpha}, tumor necrosis factor-{alpha}; TACE, TNF{alpha}-converting enzyme; TAPI-2, TNF{alpha} protease inhibitor-2; HB-EGF, heparin-binding epidermal growth factor-like growth factor; PMA, phorbol 12-myristate 13-acetate; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; TIMP, tissue inhibitor of metalloproteinase; CHO, chinese hamster ovary; SEAP, secreted human placental alkaline phosphatase; HUVEC, human umbilical vein endothelial cells; mAb, monoclonal antibody. Back


   ACKNOWLEDGMENTS
 
We thank Henry Bourne for thoughtful comments on the article.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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