AC-55541 [N-[[1-(3-bromo-phenyl)-eth-(E)-ylidene-hydrazinocarbonyl]-(4-oxo-3,4-dihydro-phthalazin-1-yl)-methyl]-benzamide] and AC-264613 [2-oxo-4-phenylpyrrolidine-3-carboxylic acid [1-(3-bromo-phenyl)-(E/Z)-ethylidene]-hydrazide] are the first two small-molecule agonists described for the G protein–coupled receptor protease-activated receptor 2 (PAR2), but whether they activate PAR2 through a similar mechanism as its tethered peptide ligand or soluble peptide mimetics of its tethered peptide ligand is unclear. Extracellular loop 2 (ECL2) has been shown to play a critical role in the activation mechanism of PAR2. Therefore, we constructed a series of PAR2 receptors mutated in ECL2, including a previously described polymorphic variant of PAR2 (F240S), and compared AC-55541 and AC-264613 to SLIGRL and a potent analog of SLIGRL called 2-furoyl LIGRLO in a series of functional assays, including cellular proliferation, phosphatidylinositol hydrolysis, and β-arrestin recruitment assays. Surprisingly, receptors with the F240S mutation were constitutively active in all functional assays tested. Furthermore, AC-55541 and AC-264613 were potentiated over 30-fold at the receptors with the F240S mutation, whereas SLIGRL and 2-furoyl LIGRLO were much less affected. In contrast, mutagenesis of charged residues in ECL2 confirmed their important role in the actions of peptide agonists of PAR2, whereas these mutations did not significantly affect activation of PAR2 by AC-55541 or AC-264613. These results suggest that F240S PAR2 receptors may be useful in screens to detect novel small-molecule PAR2 modulators and that further work on the biological importance of the F240S PAR2 variant is warranted.
Protease-activated receptors (PARs) are a family of four G protein–coupled receptors (GPCRs; PAR1, PAR2, PAR3, and PAR4) that are self-activated by tethered peptide ligands exposed by proteolytic cleavage of the extracellular amino terminus. PAR1, PAR3, and PAR4 are activated by thrombin, whereas PAR2 and, to a lesser degree, PAR4 are activated by trypsin. In addition, PAR2 can be proteolytically activated by a variety of other substances, including tryptase, factor Xa/tissue factor/factor VIIa, and the dust mite allergens Der p3 and Der p9, among others. Exposure to soluble synthetic peptides matching or approximating the sequences of their cognate tethered ligands also activates PAR1, PAR2, and PAR4, thus mimicking the effects of activating proteases (for review, see Ramachandran and Hollenberg, 2008).
Recently, we reported on the discovery and characterization of the first small-molecule PAR2 agonists AC-55541 [N-[[1-(3-bromo-phenyl)-eth-(E)-ylidene-hydrazinocarbonyl]-(4-oxo-3,4-dihydro-phthalazin-1-yl)-methyl]-benzamide] and AC-264613 [2-oxo-4-phenylpyrrolidine-3-carboxylic acid [1-(3-bromo-phenyl)-(E/Z)-ethylidene]-hydrazide] (Gardell et al., 2008; Seitzberg et al., 2008). Both compounds displayed high potency, efficacy, and selectivity for PAR2 over the other PAR subtypes in a variety of functional assays, including cellular proliferation, calcium mobilization, and phosphatidylinositol (PI) hydrolysis assays. Both compounds stimulated internalization of PAR2 receptors, and both compounds elicited hyperalgesic and proinflammatory effects in vivo.
The importance of extracellular loop 2 (ECL2) in the mechanism of PAR2 activation was previously demonstrated in a series of elegant experiments using mutant forms of PAR2 in which mutations in ECL2 altered the interactions of either the proteolytically exposed tethered ligand or the soluble peptide SLIGRL with PAR2 receptors (Al-Ani et al., 2002). Specifically, based on charge swapping experiments between the receptor and the peptide, it was shown that ionic interactions between negatively charged residues in ECL2 and positively charged residues in SLIGRL or the proteolytically exposed tethered ligand help mediate binding and receptor activation. Besides these artificial receptor mutations, a polymorphic variant of PAR2 receptors containing the ECL2 mutation F240S was previously described that also affected agonist activation of PAR2 (Compton et al., 2000).
In this study, we explored the interactions of AC-55541 and AC-264613 with PAR2 receptors mutated in ECL2 at E232, Q233, and F240. In contrast to SLIGRL or the potent SLIGRL analog 2-furoyl LIGRLO (McGuire et al., 2004), PAR2 receptor activation by AC-55541 and AC-264613 was hardly affected by mutations at E232 and Q233 but was massively potentiated by the F240S mutation. In addition, we observed that PAR2 F240S receptors were constitutively active in multiple functional endpoints, further highlighting the importance of ECL2 in the activation mechanism of PAR2 receptors.
Materials and Methods
NIH 3T3 cells (CRL 1658) and human embryonic kidney 293T cells (HEK-293T, CRL 11268) were purchased from American Tissue Culture Collection (Manassas, VA). O-Nitrophenyl-β-d-galactopyranoside and Nonidet P-40 were from Sigma-Aldrich (St. Louis, MO). The tissue culture medium used was Dulbecco’s modified Eagle’s medium (Gibco-BRL, Grand Island, NY). The 96-well tissue culture dishes were from Thermo Fisher Scientific (Pittsburgh, PA). Hanks’ balanced salt solution without magnesium chloride, magnesium sulfate, and calcium chloride and trypsin-EDTA were all from Gibco-BRL.
All compounds for in vitro studies were solubilized as 10 mM stock solutions in either water or dimethylsulfoxide. Working dilutions were made in the assay buffer appropriate to the specific functional assay. AC-55541 and AC-264613 were synthesized at ACADIA Pharmaceuticals Inc. (San Diego, CA) as described previously (Seitzberg et al., 2008). SLIGRL (Ser-Leu-Ile-Gly-Arg-Leu-NH2) and 2-furoyl LIGRLO (2-furoyl-Leu-Ile-Gly-Arg-Leu-Orn-NH2) were obtained from Tocris (Bristol, UK).
NIH 3T3 cells were incubated at 37°C in a humidified atmosphere (5% CO2) in Dulbecco's modified Eagle’s medium supplemented with 4500 mg/l glucose, 4 nM l-glutamine, 50 U/ml penicillin G, 50 U/ml streptomycin (HyClone; Thermo Fisher Scientific, Logan, UT), and 10% calf serum. HEK-293T cells were incubated at 37°C in a humidified atmosphere (5% CO2) in Dulbecco’s modified Eagle’s tissue culture medium with the same supplements used for NIH 3T3 cells with the exception of 10% fetal calf serum, which was used instead of calf serum.
The human PAR2 wild-type and S37P receptors were described previously (Gardell et al., 2008). The Q233E, E232R, E232R/Q233R, F240S, and F240S/S37P receptors were made with the wild-type and S37P receptors as templates using QuikChange Mutagenesis (Stratagene, a division of Agilent Technologies, Santa Clara, CA) according to the manufacturer’s instructions. All clones were sequence verified before use.
Cellular Proliferation Assays.
Receptor Selection and Amplification Technology (R-SAT) assays were performed in NIH 3T3 cells as described previously (Gardell et al., 2008).
Phosphatidylinositol Hydrolysis Assays.
PI hydrolysis assays were performed as previously described (Brandish et al., 2003) with the following modifications: HEK-293T cells (6 million) were grown overnight in a 10-cm dish to 60–80% confluence and transfected with 10 µg of the indicated receptors and 30 μl of FuGENE HD according to the manufacturer’s instructions (Promega, Madison, WI). At 18 hours post-transfection, the cells were harvested with a disposable cell lifter, seeded, and labeled overnight with 2-[3H]myoinositol (0.2 μCi/0.1 ml per well; PerkinElmer Life Analytical Sciences, Waltham, MA) into 96-well plates. Cells were incubated for 45 minutes in the assay buffer with the indicated concentrations of freshly diluted ligands. The reaction was stopped by dumping the medium and adding 50 μl/well 100 mM formic acid. Eighty microliters of preloaded RNA binding Ysi-SPA beads (GE Healthcare, Buckinghamshire, UK) diluted 1:8 was added to each well of a 96-well PicoPlate-96 (PerkinElmer, Shelton, CT) followed by 20 μl/well of the formic acid extracts. The plates were sealed and incubated for 1 hour with shaking. The plates were allowed to settle for 1 hour before counting on a TopCount NXT scintillation counter.
β-Arrestin Recruitment Bioluminescence Resonance Energy Transfer Assays.
β-Arrestin recruitment assays were performed using HEK-293T cells transfected with 20 µg of β-arrestin coupled to green fluorescent protein and 3.3 µg of the indicated PAR2 receptors coupled to Renilla luciferase as described previously (Burstein et al., 2011).
Receptor Internalization Assays.
Receptor internalization images were obtained in HEK-293 cells as described previously (Gardell et al., 2008).
Agonist curves from R-SAT, PI hydrolysis, and β-arrestin recruitment experiments were fitted to a sigmoidal dose-response function: Y = B + (T − B)/(1 + 10^(log EC50 − log X)), where Y is the response, B is the baseline, T is the top or maximum response, and X is the concentration of ligand. Data analysis was performed using GraphPad Prism version 4.0 (San Diego, CA).
ECL2 has been implicated as an important functional domain in the activation mechanism of the PAR2 receptor (Al-Ani et al., 2002). We previously described the discovery and characterization of AC-55541 and AC-264613, the first small-molecule agonists of the PAR2 receptor (Gardell et al., 2008; Seitzberg et al., 2008). We introduced a series of mutations located in ECL2 of the PAR2 receptor to investigate their effects on the agonist activity of AC-55541 and AC-264613 on PAR2 (see Fig. 1) compared with the soluble tethered peptide SLIGRL or a more potent analog of SLIGRL called 2-furoyl LIGRLO (McGuire et al., 2004).
Three functional outcomes were used to assess receptor signaling: PI hydrolysis, β-arrestin recruitment, and cellular proliferation using the proprietary functional assay R-SAT (Burstein et al., 2006, 2011; Gardell et al., 2008). Prior to testing the agonist activity of the small-molecule and peptide ligands described earlier, we expressed all of the mutant PAR2 receptors and assessed their constitutive activity in these three readouts. As shown in Fig. 2, there was a pronounced increase in basal, or constitutive activity, in PAR2 receptors carrying the F240S mutation in all three functional assays. The constitutive activity was not due to proteolytic activation of the receptors by proteases in the tissue culture media because it occurred even in the presence of the S37P mutation, which blocks proteolytic activation of PAR2 receptors (Al-Ani and Hollenberg, 2003). In contrast, there was no significant increase in constitutive activity of PAR2 receptors carrying the S37P mutation alone, or E232R, Q233E, or E232R/Q233R mutations compared with wild-type (WT) PAR2 receptors. There was no significant difference between WT PAR2 receptors and any of these PAR2 mutants in their maximal responses to SLIGRL, indicating that, although their levels of constitutive activity varied, their capacity for agonist activation and signal transduction was similar. As we reported previously (Gardell et al., 2008), there was no agonist response in any of these functional assays in the absence of transfected PAR2 receptors. Differences in the intracellular localization were noted; WT and S37P receptors were predominantly localized along the periphery, in the plasma membrane of the cells, whereas receptors carrying the F240S or S37P/F240S mutations were observed as punctuate clusters located in the cytoplasm, suggesting they were constitutively internalized.
Full concentration response curves for AC-55541, AC-264613, SLIGRL, and 2-furoyl LIGRLO at all of these PAR2 mutants were obtained in the cellular proliferation assay (Fig. 3; Table 1). Compared with WT and S37P receptors, the potencies of AC-55541 and AC-264613 were increased 12- to 18-fold and 26- to 34-fold at the F240S and S37P/F240S receptors, respectively. Relatively speaking, the potencies of SLIGRL and 2-furoyl LIGRLO were only slightly affected by the F240S and S37P/F240S mutations. However, consistently, the basal activity (denoted ND for no drug) was increased for the F240S and S37P/F240S receptors.
To rule out that increased expression of the F240S receptors accounted for the increased potencies of AC-55541 and AC-264613, or the increased constitutive activity of PAR2, we estimated the expression levels of the S37P/F240S receptors compared with S37P receptors by transfecting EYFP-tagged receptors and measuring the level of fluorescence in the cells. We observed that the tagged S37P/F240S receptors were expressed at 104 ± 16% of the level of the tagged S37P receptors. In addition, we confirmed that the functional properties of these tagged receptors were intact by performing cellular proliferation assays with them. The potency of AC-55541 was increased 20-fold at tagged S37P/F240S receptors compared with S37P receptors (910 vs. 46 nM), with an 8-fold response and a 5-fold increase in basal activity, very similar to results obtained with the untagged receptors (Table 1). Thus, the functional differences in cellular proliferation assays between S37P and S37P/F240S receptors are not due to differences in expression levels.
At PAR2 receptors carrying E232R, Q233E, or E232R/Q233R mutations, the opposite effects were seen (Fig. 3, D–G; Table 1). The potencies of AC-55541 and AC-264613 were not significantly affected by these mutations. In contrast, the potencies of SLIGRL and 2-furoyl LIGRLO were increased by the Q233E mutation, which increases the net negative charge of ECL2 and thus may strengthen its interactions with these peptide ligands, and decreased by the E232R/Q233R mutation, which increases the net positive charge of ECL2, thus weakening its interactions with these peptide ligands. These results for SLIGRL and 2-furoyl LIGRLO are consistent with a previous report demonstrating that ionic interactions with ECL2 contribute to SLIGRL activation of PAR2 (Al-Ani et al., 2002).
The effect of the F240S mutation on the potencies of AC-55541 and AC-264613 was so pronounced in the cellular proliferation assays that we tested these compounds at S37P and S37P/F240S PAR2 receptors in PI hydrolysis and β-arrestin recruitment assays. In the PI hydrolysis assays, virtually the same results were obtained. The potencies of AC-55541 and AC-264613 were increased 26- and 30-fold, respectively, at the S37P/F240S mutant compared with the S37P mutant, whereas the potency of 2-furoyl LIGRLO was only increased 2-fold (Fig. 4; Table 2). The characteristic constitutive activity of the receptors bearing the F240S mutation was evident in all cases.
In addition to G protein activation, β-arrestin recruitment is another major signal transduction pathway used by G protein–coupled receptors. We used bioluminescence resonance energy transfer assays using Renilla luciferase–tagged PAR2 receptors and green fluorescent protein–tagged β-arrestin 2 (see Materials and Methods) to examine the effect of the F240S mutation on this pathway. Very similar results to the results in the cellular proliferation and PI hydrolysis assays were seen (Fig. 5; Table 2). The potency of AC-264613 was increased 68-fold at the S37P/F240S receptors compared with the S37P receptors. The agonist actions of AC-55541 also appeared to be potentiated by the F240S mutation, as a maximal response of more than 100% was reached at 1 µM concentrations at the S37P/F240S receptors, whereas the response at 1 µM was less than 30% at the S37P receptors. Due to interference of AC-55541 with the Renilla luciferase signal, it was not possible to test concentrations greater than 3 µM (unpublished observations). In contrast, the potency of 2-furoyl LIGRLO was not significantly different between the two receptors, and its maximal response was decreased at the S37P/F240S receptors. There was a small but reproducible increase in constitutive activity in the β-arrestin recruitment assays at the S37P/F240S receptors compared with the S37P receptors. The luminescence signal from the Renilla luciferase–tagged S37P/F240S receptors was 93 ± 11% of the level of the Renilla luciferase–tagged S37P receptors. Thus, the functional differences in β-arrestin recruitment assays between the S37P and S37P/F240S receptors are not due to differences in expression levels.
We have conducted a structure-activity analysis of the agonist actions of the small-molecule PAR2 agonists AC-55541 and AC-264613. We constructed a series of mutations in ECL2, a region of PAR2 receptors known to play an important role in agonist activation of PAR2 receptors, either by its tethered ligand or by soluble peptide mimetics. We observed that mutations previously shown to potentiate or weaken interactions of soluble peptide mimetics had little or no effect on activation of PAR2 receptors by either AC-55541 or AC-264613. In contrast, the polymorphic variant F240S massively potentiated the actions of both AC-55541 and AC-264613, and constitutively activated PAR2.
ECL2 was previously shown to play a crucial role in the activation mechanism of PAR2 receptors (Al-Ani et al., 2002). Recently, the three-dimensional structures of several nonrhodopsin GPCRs have been reported, including a comparison of agonist- and antagonist-bound receptors, which indicate an important role for TM5 in mediating agonist-induced conformational changes (see Hulme, 2013 for review). Whether this mechanism applies to the PAR2 receptor is not known; however, it is conceivable that the F240S mutation, which is located at the junction of ECL2 and the top of TM5, may influence the conformation changes of PAR2 receptors undergoing activation through such a mechanism. Generally, constitutively activating mutations are believed to destabilize inactive conformations of GPCRs by reducing hindrances normally released by agonist activation (Kjelsberg et al., 1992; Spalding et al., 1997; Spalding and Burstein, 2006; Hulme, 2013). For PAR2 F240S mutant receptors, replacement of a large hydrophobic residue with a small polar residue may disrupt interactions important for stabilizing PAR2 receptors in an inactive conformation.
Several different mechanisms could account for the dramatic and compound-specific potentiation of AC-55541 and AC-264613 at the F240S PAR2 receptors; however, regardless of the mechanism, one potential outcome of these studies is the use of this mutant for further efforts to discover novel small-molecule PAR2 modulators. High-throughput screens using F240S receptors could potentially detect chemo types that might otherwise be missed at WT PAR2 receptors.
The potentiation of agonists is often observed in conjunction with increased constitutive activity of a receptor (Spalding et al., 1997). One possible explanation for the selective potentiation of the AC compounds could be superior access of the AC compounds to internalized receptors which presumably cannot be reached by the peptide agonists. This possibility is unlikely because 1) the peptide agonists do not lose potency at F240S receptors; they actually gain ~2- to 4-fold potency at all three functional assays; and 2) one has to assume that that the entire receptor population (internalized receptors and receptors located at the plasma membrane) contribute equally to the functional endpoints measured. This is almost certainly not the case. Another possibility is that the AC compounds interact allosterically with PAR2 receptors. We recently described M1 muscarinic receptor agonists that interact allosterically with M1 receptors, and are dramatically potentiated, in a compound-specific manner by certain point mutations (Spalding et al., 2006). One significant advantage of allosteric ligands compared with orthosteric ligands is that allosteric ligands interact with nonconserved amino acid residues, providing a basis for greatly increased receptor-subtype selectivity (Jensen and Spalding, 2004; Gao and Jacobson, 2013). This is certainly true for AC-55541 and AC-264613, which show no activity at the other PAR receptor subtypes or a variety of other GPCRs (Gardell et al., 2008). We do not have evidence that AC-55541 or AC-264613 bind PAR2 receptors simultaneously with its tethered ligand or peptide mimetics, and thus cannot say whether they are allosteric agonists of PAR2. However, the implications of these results are that it might be possible to make allosteric modulators of PAR2. A negative allosteric modulator that reduced PAR2 responsiveness to its tethered ligand might be particularly useful therapeutically.
The mutation F240S was previously reported to be a polymorphic variant of the PAR2 receptor with an allelic frequency of 0.084 for the serine variant in Caucasians (Compton et al., 2000). However, despite its high frequency, no phenotype was assigned to this mutation. Polymorphisms in GPCRs are relatively common, may include both activating and inactivating mutations, and in some cases affect the health status of individuals carrying the mutations (Thompson et al., 2008a,b). Given that F240S PAR2 receptors are constitutively activated, one would expect people carrying this polymorphic variant would display a strong phenotype. To date, there are no reports of altered disease susceptibility or physiology in persons bearing the F240S polymorphism. Our results suggest further investigation may be warranted to 1) confirm the existence of this polymorphic variant in other populations, and 2) determine whether it affects the health status of such persons.
The authors thank Yan Gao for excellent technical assistance and Morley Hollenburg for critical reading of the manuscript.
Participated in research design: Burstein.
Conducted experiments: Ma.
Performed data analysis: Ma, Burstein.
Wrote or contributed to the writing of the manuscript: Burstein.
- Received August 12, 2013.
- Accepted September 27, 2013.
- 2-oxo-4-phenylpyrrolidine-3-carboxylic acid [1-(3-bromo-phenyl)-(E/Z)-ethylidene]-hydrazide
- extracellular loop
- 2-furoyl LIGRLO
- G protein–coupled receptor
- human embryonic kidney 293T
- protease-activated receptor
- Receptor Selection and Amplification Technology
- transmembrane domain 5
- wild type
- Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics