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J. Biol. Chem., Vol. 275, Issue 50, 39207-39212, December 15, 2000
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From the
Received for publication, August 9, 2000
Protease-activated receptor 2 (PAR2) is a
trypsin-activated member of a family of G-protein-coupled PARs. We have
identified a polymorphic form of human PAR2
(PAR2F240S) characterized by a phenylalanine to
serine mutation at residue 240 within extracellular loop 2, with
allelic frequencies of 0.916 (Phe240) and 0.084 (Ser240) for the wild-type and mutant alleles,
respectively. Elevations in intracellular calcium were measured in
permanently transfected cell lines expressing the receptors.
PAR2F240S displayed a significant reduction in sensitivity
toward trypsin (~3.7-fold) and the PAR2-activating peptides,
SLIGKV-NH2 (~2.5-fold) and SLIGRL-NH2
(~2.8-fold), but an increased sensitivity toward the
selective PAR2 agonist,
trans-cinnamoyl-LIGRLO-NH2 (~4-fold). Increased sensitivity was also observed toward the selective PAR-1 agonist, TFLLR-NH2 (~7-fold), but not to
other PAR-1 agonists tested. Furthermore, we found that
TLIGRL-NH2 and a PAR4-derived peptide,
trans-cinnamoyl-YPGKF-NH2, were selective PAR2F240S agonists. By introducing the F240S mutation into
rat PAR2, we observed shifts in agonist potencies that mirrored the human PAR2F240S, suggesting that Phe240 is
involved in determining agonist specificity of PAR2. Finally, differences in receptor signaling were paralleled in a cell growth assay. We suggest that the distinct pharmacological profile induced by
this polymorphism will have important implications for the design of
PAR-targeted agonists/antagonists and may contribute to, or be
predictive of, an inflammatory disease.
The biological actions of the proteases thrombin and trypsin on
cells are increasingly being attributed to the activation of a novel
class of G-protein-regulated receptors termed protease-activated receptors (PARs)1 (1, 2).
Proteolytic cleavage of a PAR amino-terminal exodomain unmasks a
tethered ligand that binds intramolecularly onto the body of the
receptor to initiate signaling. PAR1 and PAR3 are activated by thrombin
(3, 4), PAR2 is activated by trypsin and mast cell tryptase (5-7), and
PAR4 is activated by both thrombin and trypsin (8, 9). With the
exception of PAR3, synthetic peptides corresponding to the first six
amino acids of the receptor's tethered ligand can activate the PARs
(1). The peptide SLIGKV-NH2 is employed to activate human
PAR2, although the mouse and rat sequence, SLIGRL-NH2, and
the selective PAR2 agonist
trans-cinnamoyl-LIGRLO-NH2 (tc-LIGRLO-NH2) are more potent PAR2 agonist peptides
(PAR2-APs) (10). The human PAR1-AP SFLLR-NH2 is not
specific for PAR1, since it also activates human PAR2 (11, 12).
However, TFLLR-NH2 and
Ala-parafluoro-Phe-Arg-cyclohexyl-Ala-citrulline-Tyr
(Cit-NH2) are potent PAR1-APs (2-20 µM) that
selectively activate PAR1 (13, 14). The human PAR4-AP
GYPGQV-NH2 requires much higher concentrations (100-400
µM) than other PAR-APs to initiate receptor signaling (8,
9), but the peptide AYPGQV-NH2 has recently been described as a potent (20-100 µM) PAR4-AP (15). These short
receptor-activating peptides have consequently proved to be useful
tools for identifying the potential biological roles of PARs in
vivo.
PAR2 is expressed on a variety of cell types (5, 16-23), where it has
been reported to have an inflammatory role. For example, the
inflammatory mediators, tumor necrosis factor, interleukin-1, and
lipopolysaccharide, up-regulate endothelial PAR2 expression (24), while
PAR2 activation stimulates inflammatory cytokine release from
keratinocytes (21) and promotes endothelial rolling and adhesion of
leukocytes (26). Moreover, the administration of the PAR2-AP,
SLIGRL-NH2, in vivo, induces an inflammatory
response (27). The resulting edema occurs via neuronal PAR2 activation (28). Given that PAR2 agonists can initiate an inflammatory response,
polymorphisms within PAR2 that alter receptor signaling may have
important implications for disease. The mechanism whereby the tethered
ligand activates PAR2 remains to be clearly established. Elegant
studies with the closely related thrombin receptor (PAR1) have
suggested that the tethered ligand interacts with extracellular loop 2 (ECL2), indicating that this domain is important for governing agonist
specificity and receptor signaling (29-31). Recent evidence has also
implicated ECL2 of PAR2 as a critical region for agonist specificity
(32), and PAR2 function can be significantly altered by mutating the
corresponding residues in ECL2 that were found to be of importance in
PAR1 (33). Thus, natural mutations found within ECL2 may affect a
PAR's ability to respond to its respective agonists.
In the course of cloning PAR2, we discovered a PAR2 variant
(PAR2F240S) with a phenylalanine to serine mutation at
position 240 of ECL2. We sought to determine 1) the frequency of the
variant in a normal Caucasian population and 2) whether the functional characteristics of the variant receptor differed from the wild-type receptor in terms of its activation by trypsin and a variety of PAR agonists.
Peptides and Other Reagents--
All peptides were synthesized
by the Peptide Synthesis Facility (University of Calgary, Calgary,
Alberta, Canada). Peptides were prepared in 25 mM HEPES
buffer, pH 7.4, and were standardized by quantitative amino acid
analysis to confirm peptide concentration and purity. The pGEM-T-Easy
vector, Sau96I, Taq polymerase, dNTPs, MgCl2, and 10× PCR buffer were purchased from Promega
(Southampton, UK). All oligonucleotides were synthesized at Oswel
Laboratories (Southampton, UK); FCS, Dulbecco's modified Eagle's
medium, nonenzymatic cell dissociation fluid, penicillin, streptomycin,
amphotericin, sodium pyruvate, Geneticin, and PBS (without calcium and
magnesium) were from Life Technologies, Inc.; Porcine pancreatic type
IX trypsin (13-20,000 units/mg; molar concentrations were calculated on the basis of 20,000 units/mg), sulfinpyrazone, and calcium ionophore
(A23187) were from Sigma, and pcDNA3.1(+) were from Invitrogen.
Cloning of PAR2 and the PAR2 Polymorphism--
Oligonucleotides
targeted to sequences at the beginning of (1U-sense, 5'-CCA GGA GGA TGC
GGA GC-3') and the end of the PAR2 reading frame (1D-antisense, 5'-GAG
GAC CTG GAA AAC TCA ATA-3') were used to amplify the cDNA from a
human colonic cDNA library. The PCR product was cloned into a
pGEM-T Easy vector and sequenced using the dideoxynucleotide sequencing
method (34). The cDNA encoding human PAR2 was subcloned into
pcDNA3.1(+), and sequenced. To obtain a copy of PAR2 wild type
cDNA (6), site-directed mutagenesis using the QuikChange kit
(Stratagene, Cambridge, UK) was performed according to the
manufacturer's instructions. To establish whether the
Phe240 residue was of importance for PAR2 activation in rat
PAR2, we mutated T719 to C719 in a rat PAR2
clone (10). The human wild-type clone and rat PAR2F240S
receptor clones were sequenced to confirm the engineered mutations.
Restriction Fragment Length Polymorphism Analysis--
Genomic
DNA was extracted from the blood of 125 normal Caucasian individuals by
standard techniques (35). Consent was obtained from all subjects, and
ethical approval was granted by the Southampton and SouthWest Hampshire
Joint Ethics Committee. PAR2 primers corresponding to nucleotides
699-718 in extracellular loop II (T2D-sense, 5'-GCT CTT GGT GGG AGA
CAG GT-3') and nucleotides 926-949 in extracellular loop
III (T4U-antisense, 5'-GGC TCT TAA TCA GAA AAT AAT GCA-3') were used to
generate a PCR product of 250 bp from the genomic DNA samples. A
Sau96I site was designed into the T2D-sense primer and was
accomplished by replacing nucleotide 716 (T) with G (see primer
T2D above, wherein G (716) is underlined). A control Sau96I restriction site was present 100 bp from the 3'-end of the PCR product.
The PCR was performed in a Gene Amp 2400 PCR System with 200 ng of DNA,
150 ng of each primer, 0.2 mM dNTPs, 2.5 mM
MgCl2, and 1 unit of Taq polymerase in a 50-µl
reaction volume starting at 94 °C for 5 min, 35 cycles of denaturing
at 94 °C for 30 s, annealing at 55 °C for 30 s, and
extension at 72 °C for 30 s, with a final extension at 72 °C
for 5 min. The 250-bp product was digested overnight at 37 °C with 6 units/reaction of Sau96I and then run on an acrylamide gel,
stained in ethidium bromide, and observed by ultraviolet light.
Genomic DNA Sequencing--
To confirm data obtained from the
restriction fragment length polymorphism analysis, oligonucleotides
corresponding to nucleotides 389-409 in extracellular loop I
(S1D-sense, 5'-TGA AGA TTG CCT ATC ACA TAC-3') and nucleotides 926-949
in extracellular loop III (T4U-antisense, 5'-GGC TCT TAA TCA GAA AAT
AAT GCA-3') were used to amplify human genomic DNA. The 560-bp products
were purified before fluorescence-based automated cycle sequencing was
performed on an ABI 377 sequencer (ABI PRISMTM Dye Primer
Cycle Sequencing-21M13 FS and M13REV FS Ready Reaction Kits).
Cell Culture and Transfection--
Kirsten sarcoma-transformed
rat kidney epithelial cells (KNRK; American Tissue Type Culture
Collection, Manassas, VA) were selected, since they have previously
been employed to generate PAR2-expressing cell lines (5, 10).
Semiconfluent KNRK cells in 60-mm Petri dishes were transfected using
the LipofectAMINE method according to the manufacturer's protocol
(Life Technologies, Inc.). Transfected cells were subcloned in
Geneticin (0.6 mg/ml)-containing medium (Dulbecco's modified Eagle's
medium, 5% FCS, 100 µM sodium pyruvate, 100 units/ml
penicillin, 100 µg/ml streptomycin, and 250 ng/ml amphotericin B),
and clones were initially selected by their ability to produce a
calcium signal in response to trypsin and the PAR2-selective peptide
tc-NH2. To obtain permanent cell lines, cells expressing
high levels of PAR2 were isolated by FACS using the B5 anti-PAR2
polyclonal rabbit antibody (10, 36). All cell lines were propagated in
Geneticin-containing medium without the use of trypsin in 95% air, 5%
CO2 at 37 °C. The rat PAR2 cell line has been described
elsewhere (10, 33). Clones with matched expression, as assessed by FACS
analysis, were selected for functional studies. Reverse
transcriptase-PCR (as described previously (37)) was then performed
using the primers S1D and T4U on the human wild-type and
PAR2F240S cell lines, and the 560-bp products were
sequenced to confirm the single nucleotide difference (T719
to C719) between the two cell lines.
Calcium Signaling Assay--
The calcium cell-signaling assay
was performed as described previously (33). Briefly, cells at 90%
confluence in 80-cm2 flasks (Life Technologies, Inc.) were
rinsed with PBS, lifted with nonenzymatic cell dissociation fluid, and
pelleted before resuspension in 1 ml of Dulbecco's modified Eagle's
medium, 10% FCS, and 0.25 mM sulfinpyrazone. To the cells,
10 µl of 2.5 mg/ml Fluo-3 acetoxymethyl ester (Molecular Probes,
Inc., Eugene, OR) was added prior to gentle shaking for 35 min at room
temperature. Cells were then washed in PBS and resuspended in calcium
assay buffer (150 mM NaCl, 3 mM KCl, 1.5 mM CaCl2, 10 mM glucose, 20 mM HEPES, 0.25 mM sulfinpyrazone, pH 7.4).
Fluorescence measurements were performed on a Perkin-Elmer fluorescence
spectrometer 650-10S, with an excitation wavelength of 480 nm and
emission recorded at 530 nm. Cell suspensions (2 ml of 3 × 105 cells/ml) in 4-ml cuvettes were stirred with a magnetic
flea bar and maintained at 24 °C. To establish concentration-effect curves, the signal produced by the addition of a test agonist was
measured as a percentage of the fluorescence peak height yielded by the
addition of 2 µM calcium ionophore (A23187, Sigma).
Cell Proliferation Assay--
Human wild-type PAR2- or
PAR2F240S-transfected cells were seeded in six-well culture
plates at 100,000 cells/well in fresh medium and incubated for 24 h. Medium was aspirated, and cells were washed with PBS before the
addition of 2 ml of low serum medium (0.2%). Following a 24-h
incubation, test agents were introduced. The test agent trypsin, was
added in the absence of serum for 1 h, before a final
concentration of 0.2% FCS was added. At 72 h, cells were rinsed
with PBS and harvested for counting with nonenzymatic cell dissociation
fluid. Cells were counted on an improved Neubauer hemocytometer
(American Optics, Buffalo, NY).
Statistics--
Results were analyzed for statistical
significance by the paired Student's t test, taking
p < 0.05 as statistically significant.
Cloning of the Human PAR2 Polymorphic Receptor--
A comparison
of the sequence obtained for the polymorphic receptor with the
published sequence of human PAR2 (6) revealed a transition from a
thymine to a cytosine at nucleotide position 719. Sequencing of genomic
DNA from a selection of Caucasian individuals confirmed the existence
of the mutation (Fig. 1,
upper). Individuals homozygous for wild type PAR2 were
identified by a single thymine residue at position 719 (Fig. 1,
upper (a)), while individuals homozygous for
polymorphic PAR2 were identified by the presence of a cytosine at
residue 719 (Fig. 1, upper (b)). Finally,
heterozygous individuals were identified by the presence of both a
thymine and a cytosine at residue 719 (Fig. 1, upper
(c)). This mutation is predicted to result in a mutant
receptor (PAR2F240S) with an amino acid change from
Phe240 to Ser240. The site of the mutation is
approximately 6 amino acids from the fifth putative transmembrane
domain, in ECL2 (Fig. 1, lower).
Genotyping of the PAR2 Polymorphism--
Restriction fragment
length polymorphism analysis and automated DNA sequencing were employed
to determine the frequency of the polymorphism in a Caucasian
population. The frequency of the homozygous S/S genotype was found to
be relatively uncommon (Table I). The
data were found to agree with the Hardy-Weinberg equilibrium (F2 + 2FS + S2 = 1 where F2
represents homozygote F/F frequency, S2 represents
homozygote S/S frequency, and 2FS represents heterozygote F/S
frequency), indicating that the two alleles of the PAR2 gene are
segregated in a Mendelian manner.
PAR2F240S Is Less Sensitive to the PAR2 Agonists
Trypsin, SLIGKV-NH2, and SLIGRL-NH2 but More
Sensitive to tc-LIGRLO-NH2--
The concentration-effect
curves for trypsin and several selective PAR2-APs are shown in Fig.
2. For the wild-type receptor (solid lines), trypsin was 3 orders of magnitude
more potent than the PAR2-APs, SLIGKV-NH2,
tc-LIGRLO-NH2, and SLIGRL-NH2. The relative rank order of potencies was as follows: trypsin >>>>
tc-LIGRLO-NH2 PAR2F240S Displays Increased Sensitivity to the
Selective PAR1-AP TFLLR-NH2 but Not to
SFLLR-NH2 and Cit-NH2--
The concentration
effect curves for PAR1-APs are shown in Fig. 2, e and
f. For the wild-type receptor (solid
lines), the relative rank order of potencies was as follows:
SFLLR-NH2 > Cit-NH2 > TFLLR-NH2
as previously reported (13). SFLLR-NH2,
Cit-NH2, and TFLLR-NH2 stimulated wild-type
receptor activation from 5 to 200 µM, 50 to 400 µM, and 100 to 800 µM, respectively. In the
PAR2F240S receptor (dashed lines),
SFLLR-NH2 and Cit-NH2 induced a response between 5 and 100 µM and between 50 and 400 µM, respectively, yielding concentration-effect curves
essentially the same as those for the wild-type receptor (Fig.
2e and Table II). Surprisingly, TFLLR-NH2 was
over 7-fold more potent on the PAR2F240S receptor, provoking a response from 10 to 400 µM (Fig.
2f and Table II). The relative rank order of potencies for
these PAR1-APs in the PAR2F240S receptor was
SFLLR-NH2 > TFLLR-NH2 > Cit-NH2,
which differed qualitatively from those for the wild-type receptor
(above). Following desensitization of PAR2F240S by the
prior addition of the PAR2-selective peptide tc-LIGRLO-NH2
(50 µM), TFLLR-NH2 (50 µM) was
no longer active, confirming its action on PAR2F240S (data not shown). Furthermore, thrombin at 5 units/ml (a concentration shown
to be maximal for PAR1 activation (13)) failed to induce a calcium
response in both the wild-type and PAR2F240S cell lines (data not shown), indicating an absence of functional PAR1 and PAR4 in
the KNRK system.
Rat PAR2F240S Displays Differences in Agonist
Sensitivity Similar to Human PAR2F240S--
The relative
potencies for the PAR agonists tested on the rat PAR2F240S
receptor are displayed in Table II. In the rat PAR2F240S receptor, trypsin and SLIGRL-NH2 were ~2- and ~3-fold
less potent compared with the rat wild-type receptor, respectively.
Compared with the wild-type receptor, tc-LIGRLO-NH2 and
TFLLR-NH2 were ~3-fold more potent in the rat
PAR2F240S receptor.
TLIGRL-NH2 and tc-YPGKF-NH2 Selectively
Activate PAR2F240S--
In the human wild-type receptor,
TLIGRL-NH2 (50 µM, a concentration previously
demonstrated to have little effect on PAR2 (12)) showed minimal
activity (Fig. 3a), but in the
PAR2F240S receptor it provoked a response similar to that
of SFLLR-NH2 (50 µM). In the rat wild type
receptor (Fig. 3b), TLIGRL-NH2 (50 µM) stimulated a small response, but in the rat
PAR2F240S cell line, TLIGRL-NH2 (50 µM) provoked a response comparable with
SFLLR-NH2 (50 µM). SFLLR-NH2 (50 µM) equally stimulates the wild-type and PAR2F240S. In keeping with previous reports (9, 15), the PAR-4APs AYPGKF-NH2 (100 µM) and
GYPGKF-NH2 (400 µM) were without effect in
rat and human wild-type and PAR2F240S systems (data not
shown). Similarly, the PAR4-derived peptide, tc-YGPKF-NH2, was found to have no effect on either rat or human wild-type PAR2 (Fig.
3, c and d). Strikingly, tc-YGPKF-NH2
stimulated a robust response equivalent to SFLLR-NH2 in the
human PAR2F240S system and 50% of the
SFLLR-NH2 response in the rat PAR2F240S system. Following desensitization of PAR2F240S by the addition of
the PAR2-selective agonist tc-LIGRLO-NH2 (50 µM), TLIGRL-NH2 (50 µM) and
tc-YGPGKF-NH2 (100 µM) were without effect
(data not shown), confirming the selective activation of PAR2.
Furthermore, the addition of TLIGRL-NH2 (100 µM) abrogated the response caused by a submaximal dose
(20 µM) of SLIGRL-NH2 (data not shown).
Cell Growth Assay--
The ability of selected PAR2 agonists to
inhibit cell growth in the wild-type and PAR2F240S cell
lines was assessed. Test concentrations of agonists were selected on
the basis of the data shown for calcium signaling in Figs. 2 and 3.
Results are shown in Fig. 4. Trypsin was
significantly more effective at inhibiting cell growth in the wild-type
(28.5 ± 7.5%) cell line compared with the PAR2F240S
(4.9 ± 3.7%) cell line. tc-LIGRLO-NH2,
tc-YPGKF-NH2, and TLIGRL-NH2 inhibited growth
of the PAR2F240S cell line by 32.7 ± 5.6, 40.1 ± 9.7, and 52.9 ± 4.0% respectively but had little effect on
the wild-type cell line ( The main finding of this study was the discovery of a human PAR2
genetic polymorphism that displays differential activation in response
to trypsin and other PAR agonists. To our knowledge, this is the first
report of a functionally distinct polymorphic form of a
protease-activated receptor. The polymorphic receptor displayed reduced
sensitivity to trypsin, SLIGKV-NH2, and
SLIGRL-NH2 but an increase in sensitivity to the
PAR2-selective agonist tc-LIGRLO-NH2 and surprisingly also
to the PAR1-selective agonist TFLLR-NH2. In addition, we
report that TLIGRL-NH2 and tc-YPGKF-NH2 could be utilized as selective agonists for the polymorphic receptor. Constructing the same Phe240 to Ser240 mutation
in rat PAR2 led to comparable findings, suggesting that Phe240 may participate directly in regulating agonist
specificity in PAR2 from a variety of species. Furthermore, differences
in receptor signaling between the wild-type and polymorphic cell lines,
in terms of elevations in intracellular calcium, were paralleled by
differences observed in a cell growth assay wherein PAR2 agonists inhibited cell growth, as has been previously reported (5). We propose
that the changes in responsiveness of the polymorphic receptor toward
trypsin and a spectrum of other agonists will have important
implications for the development of PAR2 antagonists and
protease inhibitors and for the possibility that this polymorphism may
play a role in disease.
We confirmed that the Phe240 to Ser240 mutation
in PAR2 was a true polymorphism by direct DNA sequencing and
restriction fragment length polymorphism analysis. In addition, we
characterized a population of Caucasian individuals to determine the
frequency of the polymorphism. Analysis of the data using the
Hardy-Weinberg equation indicated that the alleles are normally
distributed and are likely to be segregated in a Mendelian fashion.
Furthermore, the data suggest that individuals possessing both alleles
are uncommon, representing less than 2% of the population tested. Unfortunately, due to subject confidentiality, we were unable to
contact specific individuals of potential interest to explore their
possible phenotypes or association with disease. Clearly, in future
work, this issue deserves systematic investigation with new
populations, including those with a defined disease.
Using intracellular elevations of calcium as an indicator of receptor
activation, we observed that trypsin was substantially less potent at
provoking a response in the polymorphic compared with the wild-type
receptor. In agreement with the apparent reduction of receptor
sensitivity toward the tethered ligand revealed by trypsin was the
finding that SLIGKV-NH2 and SLIGRL-NH2 were
also less potent in activating the polymorphic receptor. In stark
contrast to the reduced sensitivity of PAR2F240S toward
trypsin, SLIGKV-NH2, and SLIGRL-NH2 was the
finding that tc-LIGRLO-NH2 displayed a marked
increase in potency for activating PAR2F240S
compared with the wild-type receptor. The main structural difference
between the tc-LIGRLO-NH2 compound and the other PAR2-APs
(including the tethered ligand) is the large N-terminal aromatic
trans-cinnamoyl group. The loss of the aromatic side chain
in the Phe240 to Ser240 mutation may allow the
trans-cinnamoyl group to dock more efficiently, thereby
inducing greater receptor activation. It is important to note that the
N-terminal trans-cinnamoyl group has played a key role in
the development of PAR1 antagonists (38). Consequently, the data
obtained with tc-LIGRLO-NH2 may provide a potential insight into the reactivity of the polymorphic receptor toward other PAR antagonists, which also possess aromatic side chains (39).
We extended our studies to look for potential shifts in potencies for
PAR1-APs in the polymorphic receptor. It is well established that the
PAR1 tethered ligand sequence, SFLLR ... can activate PAR2, while
the PAR2 peptide sequence SLIGRL-NH2 is without effect on PAR1 (11, 12). Of considerable interest was the finding that TFLLR-NH2, originally developed as a PAR1-selective agonist
(11-13), showed a marked increase in potency in activating
the polymorphic receptor, resulting in a shift in the rank order of
relative potencies between TFLLR-NH2 and
Cit-NH2. However, we noted that the PAR1-APs SFLLR-NH2 and Cit-NH2 possessed equal potency
in both the wild-type and polymorphic cell lines. These results would
suggest that the PAR1-APs SFLLR-NH2 and Cit-NH2
probably interact with ECL2 of PAR2 in a different manner compared with
the PAR2-APs. This conclusion is in keeping with previous observations
where a mutant rat PAR2, containing a 15-residue sequence in ECL2, that
was identical to PAR1, displayed reduced sensitivity to
SFLLR-NH2 compared with the wild-type receptor (33).
The reason for the apparent shift in the potency of
TFLLR-NH2 and not SFLLR-NH2 in the polymorphic
receptor is intriguing. In light of previous data obtained with the
TFLLR-NH2 peptide, showing a reduced potency in activating
PAR2 (12, 13), one would have predicted SFLLR-NH2 to be
more potent at activating the polymorphic receptor. Indeed,
substitutions of the serine residue at position 1 of
SLIGKV-NH2 and SFLLR-NH2 are not well tolerated
for successful activation of PAR2 (11, 12). Interestingly, substituting
the serine in SLIGRL-NH2 with a threonine generates a
peptide (TLIGRL-NH2) that is relatively selective for the
polymorphic receptor, having little effect on wild type PAR2 and no
action on PAR1 (12). It would appear that the polymorphic receptor can
tolerate conservative changes at position 1 of the activating peptide.
Equally striking was the activity of the PAR4-derived peptide,
tc-YPGKF-NH2, which appeared to be entirely selective for
the polymorphic receptor, compared with the wild-type PAR2. These
marked differences in PAR-AP-mediated receptor activation between the
wild-type and polymorphic receptor confirm that these two receptors are
functionally very distinct, at least in the way they recognize agonists
(and by extension, probably antagonists). It is interesting to note
that the rat PAR2F240S mutant echoed the results obtained
with the human polymorph, implying that the Phe240 residue
not only serves as an important residue in agonist specificity but
could also be involved in the underlying mechanism used for recognizing
specific agonists, including the tethered ligand. Furthermore, the
remarkable ability of the human PAR2 polymorphic receptor to be
activated by peptides that have little resemblance to its own tethered
ligand (e.g. tc-YGPKF-NH2 and
TFLLR-NH2) raises the possibility that other small peptides
found in vivo may activate this receptor, resulting in an
inappropriate tissue response.
Finally, we sought to determine whether the differences in receptor
activation observed in the calcium signaling assay might also be
reflected in the ability of the polymorphic receptor to inhibit cell
growth (5). In this regard, trypsin was less active in the polymorphic
cell line, in keeping with the data obtained with the calcium signaling
assay. In addition, tc-YGPKF-NH2,
tc-LIGRLO-NH2, and TLIGRL-NH2 failed to inhibit
growth of the wild-type receptor cell line but had a significant effect
on slowing the growth of the polymorphic receptor cell line. These data
were entirely in accord with the structure-activity relationships for
the calcium signaling by these agents. However, SLIGKV-NH2
appeared to inhibit cell growth to similar degrees in both cell lines,
suggesting that the relatively small differences in receptor activation
between the two cell lines observed with the calcium assay are such
that they cannot be observed by the less sensitive cell growth assay. Nevertheless, the data obtained from the cell growth assay demonstrated that this polymorphism significantly reduces the ability of PAR2 agonists to regulate cell growth and presumably to affect other PAR2-mediated cell responses.
Accumulating evidence suggests that PAR2 plays a key role in the
inflammatory process and a protective role in the airways (25, 27, 28,
40). Currently, little is known about the pathophysiological roles of
PAR2, and a better understanding will depend on the development of
selective PAR2 antagonists. To date, only one receptor-selective
nonpeptide antagonist has been developed for PAR1 (39), and as yet PAR2
antagonists are still unavailable. Considering the differential
activation of the polymorphic PAR2 receptor by a variety of PAR
agonists, we suggest that this receptor will need to be considered when
designing antagonists for any of the PARs cloned to date. Whether this
PAR2 polymorphism plays a role in disease has yet to be determined and
merits further study.
In conclusion, we have found a polymorphic variation of the human PAR2
receptor at amino acid 240, where Phe (wild type) or Ser can be found.
This variation results in significant differential receptor activation
by trypsin and PAR agonists. Such variation may represent a genetic
basis for interindividual differences in disease susceptibility,
phenotype, or response to therapeutic agents targeting PARs.
We are grateful to Laurie Robertson for help
with the FACS analysis and to Dr. Alan Gough for assistance with the
cloning techniques.
*
This study was supported in large by an Amgen Inc. grant,
with ancillary support from a National Asthma Campaign grant, an Alberta Heritage Foundation for Medical Research Fellowship, and an
Operating Grant from the Canadian Medical Research Council.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Endocrine Research
Group, Department of Pharmacology and Therapeutics, University of
Calgary, Faculty of Medicine, Calgary, Alberta T2N 4N1, Canada. Tel.:
403-220-7204; Fax: 403-270-0979; E-mail: compton@ucalgary.ca.
Published, JBC Papers in Press, September 19, 2000, DOI 10.1074/jbc.M007215200
The abbreviations used are:
PAR, protease-activated receptor;
PAR1-AP and PAR2-AP, PAR1 and PAR2 agonist
peptide, respectively;
Cit-NH2, Ala-parafluoro-Phe-Arg-cyclohexyl-Ala-citrulline-Tyr;
ECL2, extracellular loop 2;
PCR, polymerase chain reaction;
bp, base pairs;
KNRK, Kirsten sarcoma-transformed rat kidney epithelial cells;
FACS, fluorescence-activated cell sorting;
tc, trans-cinnamoyl.
A Polymorphic Protease-activated Receptor 2 (PAR2) Displaying
Reduced Sensitivity to Trypsin and Differential Responses to PAR
Agonists*
§¶,,
,
Immunopharmacology Group, University
Surgical Unit, Southampton General Hospital, Southampton, United
Kingdom SO16 6YD and the § Endocrine Research Group,
Department of Pharmacology and Therapeutics and Department of
Medicine, University of Calgary, Faculty of Medicine,
Calgary, Alberta T2N 4N1, Canada
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (31K):
[in a new window]
Fig. 1.
Upper, comparison of the Nucleotide
Sequences of the two human PAR2 alleles. Sequence analysis was
performed on a cohort of Caucasian subjects to confirm the existence of
the two PAR2 alleles. Subjects homozygous for 719-T (a) or
homozygous for 719-C (b) are shown. c,
heterozygous for 719-T/C (where Y represents T or C). The
codon of amino acid 240 modified by the mutation is
underlined. Lower, receptor model showing the
location of the polymorphism and alignment of the ECL2 amino acid
residues from human, mouse, and rat PAR2. Note the conservation at the
position of the Phe240 neighboring amino acid residues.
Amino acid numbers are from the human receptor.
Distribution of the PAR2 alleles in a normal Caucasian population
SLIGRL-NH2 > SLIGKV-NH2, which is in keeping with a previous report
(33). In the wild type receptor, trypsin stimulated a detectable
calcium elevation at 2 nM, reaching a near maximum response
at 100 nM. The effective response to the PAR2-AP,
tc-LIGRLO-NH2, was between 1 and 100 µM, for
SLIGKV-NH2 from 5 to 200 µM, and for
SLIGRL-NH2 from 2 to 100 µM (solid
lines, Fig. 2a-d, respectively). For the
PAR2F240S receptor (dashed lines),
the potencies of all the PAR2 agonists were significantly different
from those for the wild-type receptor. Trypsin was nearly 4-fold less
potent (Fig. 2a and Table II),
activating the PAR2F240S receptor from 5 to 500 nM. The parent tethered ligand peptide,
SLIGKV-NH2, was also less potent in this system by over
2-fold, activating the PAR2F240S receptor from 10 to 500 µM (Fig. 2b and Table II). However, in both
the wild-type and PAR2F240S receptor systems, the maximal response to SLIGKV-NH2 appeared to be lower than the
maximal response obtained with the other PAR2 agonists (~50
versus ~60% relative to calcium ionophore). Strikingly,
tc-LIGRLO-NH2 was 4-fold more potent in the
PAR2F240S receptor (Fig. 2c and Table II),
activating PAR2F240S from 0.2 to 20 µM
compared with 1-50 µM in the wild-type receptor. The
potency of SLIGRL-NH2 was reduced by a similar degree to
that of SLIGKV-NH2 (~2.5-fold), activating
PAR2F240S from 5 to 200 µM (Fig. 2 and Table
II). The relative order of potencies for these agonists in the
PAR2F240S system, which differed quantitatively from those
for the wild-type receptor, was as follows: trypsin 
tc-LIGRLO-NH2 SLIGRL-NH2 > SLIGKV-NH2.
View larger version (27K):
[in a new window]
Fig. 2.
Calcium signaling in the human wild-type PAR2
and PAR2F240S in response to trypsin, PAR2-APs, and
PAR1-APs. Concentration-effect curves are shown for both the
PAR2 agonists (trypsin (a), SLIGKV-NH2
(b), tc-LIGRLO-NH2 (c), and
SLIGRL-NH2 (d)) and for the PAR1 agonists
(SFLLR-NH2 and Cit-NH2 (e) and
TFLLR-NH2 (f)). Wild type responses are
designated by solid lines with filled
symbols, - 
-, except for Cit-NH2, signified
by -
-; PAR2F240S responses are signified by
dashed lines with open
symbols, -
-, except for Cit-NH2 signified
by --. Cells were lifted with nonenzymatic cell dissociation fluid
and loaded with Fluo-3 (22 µM) prior to incubation for 35 min at room temperature. Cells were challenged with different
concentrations of PAR agonists, and responses were monitored by
fluorescence spectrophotometry (excitation 480 nm, emission 530 nm).
Responses were normalized to the peak height obtained with 2 µM calcium ionophore. Each data point represents the
mean ± S.E. of three or four separate experiments, each composed
of measurements obtained with duplicate cell suspensions.
Ratio (polymorphic/wild) of agonist potencies between the
PAR2F240S and wild-type receptor systems
View larger version (15K):
[in a new window]
Fig. 3.
Calcium signaling in human and rat wild-type
and PAR2F240S cell lines in response to
TLIGRL-NH2 and tc-YGPKF-NH2. A
and C, responses of human wild-type PAR2 and
PAR2F240S to TLIGRL-NH2 (A) and to
tc-YGPKF-NH2 (C). B and D,
responses of rat wild-type PAR2 and PAR2F240S to
TLIGRL-NH2 (B) and to tc-YGPKF-NH2
(D). The control exposure to SFLLR-NH2 ( ) is
shown to the right of each tracing. Cells were
lifted with nonenzymatic cell dissociation fluid and loaded with Fluo-3
(22 µM) prior to incubation for 35 min at room
temperature. Cells were challenged with the agonists shown, and
responses were monitored by fluorescence spectrophotometry (excitation,
480 nm; emission, 530 nm). Responses were compared with the peak height
obtained with SFLLR-NH2 (50 µM).
4.9 ± 2.8, 7.4 ± 4.1, and 6.8 ± 5.7%, respectively). SFLLR-NH2 inhibited
growth in the wild-type and PAR2F240S cell lines by
31.6 ± 4.9 and 45.1 ± 6.1%, respectively. SLIGKV-NH2 inhibited growth by 24.1 ± 6.3 and
25.8 ± 11.3% in the wild-type and PAR2F240S cell
lines, respectively. Cell numbers in untreated wells increased from
1 × 105 to 6 × 105 over the 96-h
period of the experiment but were reduced in the presence of PAR2-APs,
without any evidence of cell death as assessed by trypan blue
exclusion.
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Fig. 4.
Inhibition of cell growth of human wild-type
and PAR2F240S cell lines in response to PAR agonists.
Semiconfluent wild-type or PAR2F240S expressing cell lines
were incubated in 0.2% serum-containing medium for 24 h before
agonists were introduced and incubated for a further 48 h (except
for trypsin, which was incubated with cells in serum-free medium for
1 h before the addition of a final concentration of 0.2% FCS).
Cells were lifted and counted on a hemocytometer. The inhibition of
growth caused by PAR agonists was expressed as a percentage increase of
cells in the treated cultures ( 
Nt) relative to the number
of cells in the control untreated cultures (Nc).
Inhibition (%) = (1 (Nt/Nc)) × 100. KV-NH2, SLIGKV-NH2;
tc-NH2, tc-LIGRLO-NH2;
SF-NH2, SFLLR-NH2;
tc-KF-NH2, tc-YGPKF-NH2;
TL-NH2, TLIGRL-NH2. Results are
expressed as the mean ± S.E. of three or four separate
experiments, each performed in triplicate. *, p < 0.05 compared with wild-type response to respective agonist.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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