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Diabetes/Endocrine (B.A.-A., K.K.H., M.D.H.) and Mucosal Inflammation (K.K.H., M.D.H.) Research Groups, Departments of Pharmacology and Therapeutics (B.A.-A., K.K.H., M.D.H.) and Medicine (K.K.H., M.D.H.), the University of Calgary Faculty of Medicine, Calgary, Alberta, Canada
Received June 27, 2003; accepted September 29, 2003
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Abstract |
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), involves the proteolytic unmasking of an amino-terminal receptor sequence (S37LIGRLDTP...) that acts as a receptor-activating tethered ligand (Vu et al., 1991; Nystedt et al., 1994). As with PAR1, PAR2 can be activated by short peptides (e.g., S1LIGRL6-NH2) based on the tethered ligand sequence. These receptor-activating peptides (PAR2APs) can mimic the activation of PAR2 by trypsin in tissues and PAR2-expressing cells (Nystedt et al., 1994; Al-Ani et al., 1995; Böhm et al., 1996; Hollenberg et al., 1997; Déry et al., 1998; Hollenberg and Compton, 2002). Although the structure-activity relationships (SARs) for PAR2 activation by synthetic peptides based on the S1LIGRL6-NH2 motif have been studied in some depth (Hollenberg et al., 1996, 1997; Kawabata et al., 1999; Maryanoff et al., 2001), there has yet to be a systematic SAR study to determine the key residues in the PAR2 revealed tethered ligand sequence that cause receptor activation. For the peptides, leucine at position 2 is essential for receptor activation, and the isoleucine at position 3 and the arginine at position 5 both contribute to peptide potency. Importantly, simply reversing the first two amino acids (L1SIGRL6-NH2) leads to a complete loss of activity, as does acylation of the amino terminus (N-acyl-S1LIGRL6-NH2). In contrast, the relative importance of these same residues in the proteolytically revealed tethered ligand has not yet been established. Furthermore, data obtained by us for PAR2 (Al-Ani et al., 1999a, 2002b) and by others for PAR1 (Blackhart et al., 2000) suggest that the soluble peptide agonists and the corresponding tethered ligand sequences seem to interact differently with the body of the receptor. Based on the apparent discrepancies between our previous SAR work with the synthetic PAR2APs and our preliminary findings with the receptor mutants with changes both in extracellular loop-2 and in the tethered ligand of PAR2 (Al-Ani et al., 2002b), we decided to explore further the SAR profile for the tethered ligand sequence itself.
We hypothesized that 1) the amino-terminal amino acids (S37L38) would be crucial for receptor activation and that 2) Ile39 and Arg41 would also contribute to receptor activation by the tethered ligand. To test these hypotheses, all of the first six amino acids at the amino terminus of the tethered ligand were replaced with alanines (Table 1). The wild-type receptor and the alanine-replacement receptor mutants were expressed in Kirsten-virus-transformed rat kidney (KNRK) cells (Al-Ani et al., 1999a), along with PAR2L37S38, having a reversal of the first two tethered ligand residues (S37L38 
L37S38). A calcium signaling assay (Al-Ani et al., 1999a; Kawabata et al., 1999) was used to assess activation of the wild-type and mutated receptors both by trypsin and by the PAR2AP S1LIGRL6-NH2. In addition, the calcium signaling assay was used to monitor, in wild-type PAR2 (PAR2wt) and in the mutant receptors, the activity of synthetic peptides having the same sequences as those of the mutated tethered-ligand sequences (e.g., A1LIGRL6-NH2, corresponding to A37LIGRL42...).
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Added to the functional evaluation of the expressed receptor mutants, the efficiency of receptor cleavage by trypsin to unmask the tethered ligand was monitored for all PAR2 variants using an antibody (SLAW-A) that recognizes the sequence released at the R36/S37 cleavage/activation site. A loss of reactivity with SLAW-A (immunocytochemistry) confirms the release of the N-terminal receptor sequence that masks the tethered ligand (Compton et al., 2001; Al-Ani et al., 2002a). Our data point to the key role played by the first two amino acids of the tethered ligand (S37L38), in contrast with other residues within the first six amino acids that play lesser roles.
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Materials and Methods |
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2 nM).
Preparation of PAR2 Constructs and their Expression in KNRK cells. As previously documented (Saifeddine et al., 1996; Al-Ani et al., 1999a,b), rat PAR2 was cloned from kidney cDNA using the primer pairs: forward primer (containing a HindIII site and Kozak sequence shown in bold), 5'-TCAAGCTTCCACCATGCGAAGTCTCAGCCTGGC-3'; reverse primer (containing SmaI site shown in bold), 5'-CCCGGGCTCAGTAGGAGGTTTTAACAC-3'. Then, the rat PAR2 cDNA, for which sequence verification was done (Sanger et al., 1977; DNA services facility at the University of Calgary) was subcloned further into the pcDNA3 mammalian expression vector (Invitrogen, Carlsbad, CA), which was used to prepare all 10 receptor mutants shown in Table 1. The receptor mutants described in Table 1 were prepared using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. In PAR2S37A, PAR2L38A, PAR2I39A, PAR2G40A, and PAR2R41A, residues S37, L38, I39, G40, and R41 were changed to A, respectively; in PAR2A37-38, PAR2A39-42, PAR2A37, 39-42, and PAR2A37-42, residues S37L38, I39G40R41L42, S37I39G40R41L42, and S37L38I39G40R41L42 were changed to A, respectively. In PAR2L37S38, residues S37L38 were reversed to become L37S38. The wild-type and PAR2 mutants in pcDNA3 were then transfected into KNRK cells (American Type Culture Collection, Manassas, VA), as described previously (Al-Ani et al., 1999a,b) to yield permanent cell lines for further study. Transfected cells (either vector alone or PAR2-containing vectors) were subcloned in G-418-containing medium (0.6 mg/ml), and PAR2-expressing cells were isolated by fluorescence-activated cell sorting (FACS) with the use of the anti-receptor SLAW-A antibody as described elsewhere for a B5 anti-PAR2 antibody used by us previously (Kong et al., 1997; Al-Ani et al., 1999a,b). The SLAW-A antiserum recognizes the PAR2 receptor sequence that is released upon proteolytic activation of the receptor (5SLAWLLG11-G30PNSKGR36-GGYGGC) (receptor antigenic sequences represented in bold; GGYGGC added for radiolabeling and cysteine-coupling). In the cell lines so isolated, >80% of the populations (flow cytometry) were found to exhibit reactivity with the SLAW-A antibody with an equivalent fluorescence intensity on a percell basis, in keeping with our previous work (Al-Ani et al., 1999a, 2002b). Cells were routinely propagated as described previously (Al-Ani et al., 1999a) in G-418 (0.6 mg/ml)-containing growth medium and were subcultured by re-suspension in calcium-free isotonic saline/EDTA solution, without the use of trypsin.
Evaluating the Cleavage of PAR2 Variants by Trypsin. PAR2 variant cell lines were grown to about 85% confluence. These clones possess an N-terminal sequence that is proximal to the receptor's cleavage/activation sequence and therefore potentially released from the cell upon cleavage of PAR2 by trypsin at site Arg36. The rabbit polyclonal antiserum (SLAW-A) mentioned above was employed to monitor the disappearance of the signal (generated by the receptor sequence up to and including residue Arg36) upon trypsin treatment of all expressed mutants, thereby confirming trypsin cleavage (Compton et al., 2001; Al-Ani et al., 2002a). In brief, KNRK cells expressing the receptor constructs were exposed to 20 units/ml trypsin (40 nM) for 5 min at room temperature, and proteolysis was terminated by the addition of 1 µg/ml soya trypsin inhibitor. Cells were then harvested by a cytospin procedure in preparation for immunocytochemical detection of receptor with SLAW-A, comparing the receptor staining observed in cells both before and after trypsin treatment with reference to the disappearance of the SLAW-A immunoreactivity observed in control wild-type PAR2-expressing KNRK cells. To quantify the disappearance of SLAW-A-reactive epitope by trypsin treatment, as monitored by immunocytochemistry, a morphometric analysis was used as described previously (Compton et al., 2001; Al-Ani and Hollenberg, 2003). In brief, several microscopic fields, comprising 200 or more fixed stained cells, were surveyed at random, and cells were scored as either SLAW-positive or SLAW-negative. The ratio of positive to negative cells in the untreated or trypsin-treated cells was then calculated. Upon trypsin treatment, a loss of over 80% of SLAW-A reactivity was routinely observed in all previous control experiments with wild-type PAR2KNRK cells. The values obtained using the immunocytochemical approach agreed with data obtained using FACS analysis to document the removal of the SLAW-A epitope by trypsin (Compton et al., 2001; Al-Ani and Hollenberg, 2003).
Calcium Signaling Assay. Measurements of trypsin and peptide-stimulated fluorescence emission (reflecting an increase in intracellular calcium from a baseline of about 30 nM to a peak of about 340 nM) were done with cells grown to about 85% confluence and disaggregated with calcium-free isotonic phosphate-buffered saline containing 0.2 mM EDTA. PAR2-transfected KNRK cells were loaded with the intracellular calcium indicator Fluo-3 (Molecular Probes Inc., Eugene, OR) at a final concentration of 22 µM (25 µg/ml) of fluo-3 acetoxymethyl ester (Kao et al., 1989; Minta et al., 1989), as described previously (Al-Ani et al., 1999a; Kawabata et al., 1999). Fluorescence measurements, reflecting elevations of intracellular calcium, were conducted at 24°C using an AMINCO-Bowman series 2 luminescence spectrometer (Spectronic Unicam, Rochester, NY), with an excitation wavelength of 480 nm and an emission recorded at 530 nm. The fluorescence signals caused by the addition of test agonists (trypsin or peptides, added to 2 ml of a cell suspension of about 3 x 105 cells/ml) were expressed as described previously (Al-Ani et al., 1999a; Kawabata et al., 1999; Compton et al., 2000), relative to the fluorescence peak height yielded by replicate cell suspensions treated with 2 µM concentrations of the ionophore A23187
[GenBank]
(Sigma Chemical). Measurements were done using three or more replicate cell suspensions derived from two or more independently grown groups of cells. To express quantitatively the sensitivity of the PAR2 tethered ligand mutants for trypsin activation, relative to the sensitivity of wild-type PAR2, a ratio was calculated (REC,T) based on the concentration of trypsin required to cause a given calcium signal in the wild-type receptor (ECWT) relative to the concentration of trypsin required to cause the equivalent calcium signal in the mutant PAR2 receptor with an altered tethered ligand sequence (ECMUTANT). Thus, for trypsin activation, REC,T = ECWT/ECMUTANT. Values of this ratio <1 denote a receptor that requires a higher concentration of trypsin to cause the same cellular response as for PAR2wt and is therefore less sensitive than the wild-type receptor. Similarly, as we have done previously (Hollenberg et al., 1997), we expressed the sensitivity of the receptors to the synthetic peptide analogs also as a ratio (REC,P) of the concentration of the wild-type peptide, SLIGRL-NH2, required for a given calcium signal (ECSLIGRL-NH2), relative to the concentration of test peptide (ECPEPTIDE) required to generate the equivalent calcium response. Data for the wild-type receptor, denoted in the text by open symbols for peptide concentration-effect curves, are shown in Fig. 4; the peptide sensitivities of the mutant receptors are denoted by closed symbols in Fig. 4. The EC values were obtained along the linear portions of the concentration-response curves, like those shown in Figs. 2 and 4. Four to six points along the concentration-response curves were used to calculate the averages for the REC,T and REC,P values. Measurements done in this manner yielded average values, for which the standard error of the mean was less than 10% of the magnitude of the average.
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80% of all cells in each line were found to express the receptor by immunocytochemical morphometric analysis (data not shown). More importantly, the responsiveness (calcium signaling) of all PAR2 variant cell lines to the PAR2AP SLIGRL-NH2 was equivalent, with comparable EC50 values (3 to 10 µM) and maximal calcium signals at 50 µM SLIGRL-NH2 that were 80% or greater than the signal generated by PAR2wt (Fig. 1 and data not shown for constructs designated by 
and 
in Table 1).
Sensitivity of the PAR2 Variants to Trypsin. Given that the different cell lines expressed an equivalent cell surface abundance of functional receptor determined by FACS analysis and responsiveness to SLIGRL-NH2, the next step was to measure their sensitivity to trypsin, reflecting the activity of the revealed tethered ligand (Fig. 2). Most striking was the complete lack of activity of trypsin in PAR2L37S38, which was otherwise fully responsive to SLIGRL-NH2 (Fig. 2 and Table 2, 
), and the essential lack of trypsin sensitivity of the construct with A37A38 substitutions (PAR2A37-38, 
; Fig. 2). In contrast, changing only the first amino acid to alanine (PAR2S37A, 
; Fig. 2) resulted in a receptor with sensitivity toward trypsin that was the same as that of the wild-type receptor (PAR2wt, 
; Fig. 2). In contrast, changing Leu38 to alanine at the second position of the tethered ligand (PAR2L38A: 
, Fig. 2) caused a 
10-fold reduction in the sensitivity to trypsin but nonetheless resulted in a receptor that could still respond to trypsin with a maximal calcium response of 75% of that observed for PAR2wt. In addition to the crucial importance of the first two amino acids for tethered ligand activity, further alanine substitutions revealed the importance of residues Ile39 and Arg41 (PAR2I39A and PAR2R41A, 
and ; Fig. 2).
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Overall, the relative potencies of trypsin, reflecting the relative activities of the sequences as tethered ligands unmasked by proteolysis, were (Fig. 2 and Table 2): SLIGRL... 
ALIGRL... SLIARL... > SLAGRL... SLIGAL... SLAAAA... > SAIGRL... >> AAAAAA...; all of the AAIGRL..., ALAAAA..., and LSIGRL... tethered ligand sequences exhibited little or no activity (Fig. 2). Quantitatively, the sensitivities of all receptor variants toward trypsin, relative to PAR2wt (REC,T values), are summarized in Table 2, expressed as a ratio (REC,T) of the concentrations of trypsin required to cause a calcium response in the wild-type receptor (ECWT) relative to the concentration of trypsin required to generate the equivalent calcium signal in the receptor mutant (ECMUTANT). Thus, REC,T = ECWT/ECMUTANT, where values <1.0 denote a receptor mutant with reduced sensitivity to trypsin.
Unmasking of Receptor Variants by Trypsin. Although all receptor variants expressed comparable amounts of functional cell surface receptors (responses to SLIGRL-NH2 and FACS), it was essential for interpreting the trypsin sensitivity data (Fig. 2) to know that all variants were similarly cleaved by trypsin at Arg36 to reveal the tethered ligand sequence. To assess the unmasking of the tethered ligand sequence in all variants, cells were first exposed to trypsin for 5 min at a trypsin concentration (40 nM; 20 units/ml) sufficient to expose the tethered ligand and generate a maximum calcium signal in PAR2wt. Cleavage was monitored as outlined under Materials and Methods, with the SLAW-A antibody that detects only the receptor sequence removed by trypsin. As shown in Fig. 3, trypsin was able efficiently to remove the epitope detected by SLAW-A from the wild-type receptor that is fully activated by trypsin, as well as from receptor variants that showed either reduced sensitivity (PAR2A39-42) or no activity (PAR2L37S38) upon trypsin activation. A similar removal of the epitope visualized by the SLAW-A antibody was also observed for all mutant PAR2 cell lines (not shown). Morphometric analysis of the fixed stained cells revealed that, as for PAR2wt, brief trypsin treatment eliminated SLAW-A reactivity from 80% or more of all of the mutant receptor-bearing cells. Comparable results were obtained using FACS analysis of the trypsin-treated cells (not shown). Thus, trypsin treatment caused an equivalent cleavage and exposure of the tethered ligand in all receptor mutants.
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Activity of Tethered Ligand Sequences as Soluble Peptides. Although in previous work, we and others had obtained structure-activity data for alanine substitutions in the receptor-selective PAR2-activating peptide sequence, S1LIGRL6-NH2 (Hollenberg et al., 1996, 1997; Maryanoff et al., 2001), it was necessary in the present study to evaluate again the activity of the synthetic peptides corresponding to the mutated tethered ligand sequences not only in PAR2wt but also in the receptor mutants possessing the corresponding tethered ligand sequence with the `alanine walk' mutations. Thus, as outlined in Table 1 and Fig. 4, nine synthetic peptides, SLIGRL-NH2 (wild-type sequence), ALIGRL-NH2, SAIGRL-NH2, SLAGRL-NH2, SLIARL-NH2, SLIGAL-NH2, AAIGRL-NH2, SLAAAA-NH2, and LSIGRL-NH2 were tested for activity (calcium signal) in both PAR2wt and in most of the receptor mutants having the cognate tethered ligand sequence. Because neither AAIGRL-NH2 nor SLAAAA-NH2 was found to be active in the calcium signaling assay (below), the peptides ALAAAA-NH2 and AAAAAA-NH2 were presumed to be inactive and were not tested in the interests of economy. As shown in Fig. 4, at concentrations in the range of 200 to 400 µM, the peptides SAIGRL-NH2 (, 
), AAIGRL-NH2 (, 
), SLAAAA-NH2 (
, 
), and LSIGRL-NH2 (, 
) were completely inactive both in PAR2wt and in the receptor mutants possessing the same sequence as the mutated tethered ligand. In contrast, the other peptide analogs displayed relative potencies that clustered in three groups, with EC50 values in the ranges of 3, 35, and 120 µM, as summarized in the next paragraph.
The relative order of potencies of the peptides for activating the receptor (Fig. 4 and Table 2) was: SLIGRL-NH2 SLIARL-NH2 >> ALIGRL-NH2 >> SLAGRL-NH2 SLIGAL-NH2, in contrast with the inactive peptides, SAIGRL-NH2, AAIGRL-NH2, SLAAAA-NH2, and LSIGRL-NH2. The relative potencies of these different sequences for activating PAR2 in relation to the activity of SLIGRL-NH2 were expressed quantitatively, as we have done previously (Hollenberg et al., 1997), as a ratio (REC,P) of the concentration of SLIGRL-NH2 required to cause a given calcium signal ECSLIGRL-NH2, relative to the concentration of the peptide analog ECPEPTIDE required to generate the equivalent calcium response in either the wild-type receptor (PAR2wt) or in the corresponding receptor mutant (i.e., REC,P = ECSLIGR-NH2 / ECPEPTIDE: Table 2).
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To interpret the sensitivity to trypsin (calcium signal) as reflecting the activity of the tethered ligand, it was important to establish two key criteria: 1) that the receptor mutants were expressing an equivalent abundance of functional cell surface receptor and 2) that the tethered ligand in all of the receptor variants was unmasked by trypsin with comparable efficiency. Our data indicate that equivalent densities of functional receptor were indeed expressed in all cell lines (FACS analysis and comparable sensitivities to the PAR2-activating peptide, SLIGRL-NH2). Furthermore, the immunocytochemical analysis of trypsin-treated receptor-expressing cells revealed an equivalent removal of the sequence N-terminal to Arg36 that is visualized by SLAW-A. We had already identified the R36S37 site as the principle target for trypsin cleavage in the expressed receptor, resulting in the removal of the N-terminal epitope visualized by the SLAW-A antibody (Al-Ani and Hollenberg, 2003). Thus, both of the principal criteria required to interpret the trypsin sensitivity data were met, and the simple presence or absence of signaling generated by trypsin can be taken clearly to reflect the activity or lack thereof of a given tethered ligand sequence. That said, interpreting the relative activities of trypsin as reflecting the relative activities of the revealed tethered ligands in those receptor mutants that did yield calcium signals requires a further assumption that must be taken into account.
The assumption that must be made to use the REC,T values to reflect the relative activities of the tethered ligand mutants is that the rate of trypsin cleavage must be the same for all receptor mutants and that this rate must not be limiting for generating a calcium signal. We were able with the immunocytochemical/morphometric approach to determine successfully that the extent of trypsin cleavage was the same for all mutants, within the time frame of signaling (5 min); unfortunately, however, we could not accurately measure the precise rate constants for cleavage for each construct because of the technical limitations of our measurements of SLAW-A epitope removal. That said, the finding of equivalent cleavage of all PAR2 variants within 5 min strongly supports the assumption that the cleavage rates were comparable. Moreover, the clustering of the REC,T values for 1) PAR2wt, PAR2S37A, and PAR2G40A, and 2) PAR2I39A, PAR2R41A, and PAR2A39-42 (Fig. 2 and Table 2) with mutations either at or downstream of the trypsin cleavage/activation site supports the hypothesis that the trypsin cleavage rate is not a factor in interpreting the data.
Given that the two key criteria for interpreting the relative trypsin sensitivities were essentially met, we used the REC,T values (Table 2) to reflect the relative activities of the revealed tethered ligands. There were major discrepancies between the SARs for activation of the receptor by the tethered ligand sequences (Fig. 2 and Table 2) as opposed to the SARs for the soluble PAR-APs (Fig. 4 and Table 2). For instance, the sequences S37LAAAA and S37AIGRL... were quite active as tethered ligands (, ; Fig. 2), whereas the peptides S1LAAAA6-NH2 and S1AIGRL6-NH2 were devoid of activity (, , , ; Fig. 4). Furthermore, the tethered ligand sequence, A37LIGRL..., was as active as the wild-type sequence, whereas the activity of the peptide, A1LIGRL6-NH2, was considerably reduced (about 10-fold) compared with SLIGRL-NH2 (Fig. 4 and Table 2). Thus, in keeping with previous results obtained by us and by others (Blackhart et al., 2000; Al-Ani et al., 2002b), our new data point out more emphatically that the SAR data obtained for activation of the proteinase-activated receptors only by the synthetic peptides cannot be used as a basis for understanding the tethered ligand mechanism.
In our previous study (Al-Ani et al., 2002b), we focused primarily on potential interactions between the fifth residue of the revealed tethered ligand (Arg41) and acidic residues in extracellular loop-2. Our data provided evidence against such an interaction but did not at all establish the key tethered ligand residues essential for receptor activation. Clearly, simply reversing the first two residues of the revealed tethered ligand (PAR2L37S38) or replacing the first two residues with alanine (PAR2A37-38), leaving the other tethered ligand residues unchanged, substantially reduced or completely abrogated the ability of the revealed tethered ligand to activate the receptor (Fig. 2, and ). However, with all other amino acids except the Ser37 and Leu38 residues changed to alanines (PAR2A39-42), the revealed tethered ligand (S37LAAAA...) was still able to cause a substantial activation of the receptor (Fig. 2, ). The interaction of the S37L38 motif with the body of the receptor would therefore seem to be both sufficient and necessary to activate the receptor. Nonetheless, substituting alanine for serine at the N terminus of the revealed tethered ligand (PAR2S37A) led to a sequence with an activation profile equivalent to that of the wild-type tethered ligand (compare and in Fig. 2). Other amino acids that can substitute for the S37L38 motif of the revealed tethered ligand to yield full receptor activation remain to be determined. In this regard, substitution of the Leu38 with alanine (PAR2L38A, ; Fig. 2) led to a considerable loss of activity of the tethered ligand. Based on our data, one can suggest that hydrophobic residues at positions 2 and 3 of the tethered ligand may interact in a complementary pocket of the remainder of the receptor to trigger signaling.
In contrast with the discrepancies already mentioned, the SAR data for the tethered ligand sequences do parallel, to some degree, the SAR profile for the PAR2-activating peptides (Hollenberg et al., 1996, 1997; Maryanoff et al., 2001). For instance, neither the peptide LSIGRL-NH2 nor the tethered ligand sequence L37SIGRL... was able to cause receptor activation. Furthermore, the previous SAR data for the synthetic peptides pointing to the importance of the isoleucine at the third position and the arginine at the fifth position (Hollenberg et al., 1996, 1997; Maryanoff et al., 2001), are mirrored by the reduced activity of S37LAGRL... and S37LIGAL... as tethered ligands. Where concordant, the SAR data for the tethered ligand mutants and the soluble PAR2APs add support to our hypothesis that Ile39 and Arg41 play important roles in the tethered ligand activation process. This information bears directly on the future development of much needed PAR2 antagonists.
It was unexpected that the tethered ligand sequence A37LAAAA... was essentially inactive, given that sequences S37LAAAA... and A37LIGRL... both showed activity. Furthermore, it was surprising that the tethered sequence A37AAAAA... was able to activate the receptor (Fig. 2, 
), albeit with a substantially lower activity than that of the sequence, S37LAAAA... (Fig. 2, ). It seems that although there are specific steric requirements for an efficient activation of the receptor (the S37L38 motif), there may also be `negative' constraints built into the tethered ligand that can be removed by the homogeneous replacement of all six tethered ligand residues by alanine.
Taken together, our data highlight the primary importance of the first two tethered ligand amino acids, SL, as critical for receptor activation. This conclusion could not have been reached based on the SAR data obtained with the soluble PAR2Aps alone. Furthermore, our study indicates the contributions (albeit secondary) of the third and fifth (Ile39 and Arg41) residues for tethered ligand activity. Thus, for the design of potential PAR2 antagonists, the pharmacophores of the SL motif would seem to be paramount, in concert with the Leu39 and Arg41 side chains. That said, the activity of PAR2A37-42 with alanine replacements at all six tethered ligand residues should sound a cautionary note, suggesting that the proteolytic exposure of the tethered ligand may remove a prior structural constraint that enables the SL motif, to trigger signaling efficiently. It will be of considerable interest in future work to determine whether the first two amino acids of the PAR1 tethered ligand are similarly critical for receptor activation.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: PAR, proteinase-activated receptor; PAR-APs, proteinase-activated receptor-activating peptides; SAR, structure-activity relationship; KNRK, Kirsten virus-transformed rat kidney cells; PAR2wt, wild-type rat proteinase-activated receptor 2; SLAW-A, SLAWLLGGPNSKGR; FACS, fluorescence-activated cell sorting; A23187 [GenBank] , calcimycin.
1 Current address: Department of Reproductive and Vascular Biology, The Medical School, University of Birmingham, Edgbaston, Birmingham, United Kingdom B15 2TT. 
Address correspondence to: Morley D. Hollenberg, Department of Pharmacology and Therapeutics, University of Calgary Faculty of Medicine, 3330 Hospital Dr. N.W., Calgary, AB Canada T2N 4N1. E-mail: mhollenb{at}ucalgary.ca
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) or the sequences of the corresponding mutated tethered ligands. Increases in intracellular calcium were monitored, as outlined in the legend to 
, SLIARL-NH2; 
, ALIGRL-NH2; 
, SLIGAL-NH2; 
, SLAGRL-NH2; 
, PAR2A37,39-42; 
