- Methodology article
- Open Access
Optical biosensor differentiates signaling of endogenous PAR1 and PAR2 in A431 cells
© Fang and Ferrie; licensee BioMed Central Ltd. 2007
Received: 01 December 2006
Accepted: 22 June 2007
Published: 22 June 2007
Protease activated receptors (PARs) consist of a family of four G protein-coupled receptors. Many types of cells express several PARs, whose physiological significance is mostly unknown.
Here, we show that non-invasive resonant waveguide grating (RWG) biosensor differentiates signaling of endogenous protease activated receptor subtype 1 (PAR1) and 2 (PAR2) in human epidermoid carcinoma A431 cells. The biosensor directly measures dynamic mass redistribution (DMR) resulted from ligand-induced receptor activation in adherent cells. In A431, both PAR1 and PAR2 agonists, but neither PAR3 nor PAR4 agonists, trigger dose-dependent Ca2+ mobilization as well as Gq-type DMR signals. Both Ca2+ flux and DMR signals display comparable desensitization patterns upon repeated stimulation with different combinations of agonists. However, PAR1 and PAR2 exhibit distinct kinetics of receptor re-sensitization. Furthermore, both trypsin- and thrombin-induced Ca2+ flux signals show almost identical dependence on cell surface cholesterol level, but their corresponding DMR signals present different sensitivities.
Optical biosensor provides an alternative readout for examining receptor activation under physiologically relevant conditions, and differentiates the signaling of endogenous PAR1 and PAR2 in A431.
Protease activated receptors (PARs) comprise a family of G protein-coupled receptors (GPCRs) which to date include PAR1, PAR2, PAR3 and PAR4 [1–5]. Instead of being activated through reversible ligand binding, PARs utilize a unique proteolytic mechanism for activation [6–8]. Serine proteases such as thrombin and trypsin site-specifically cleave the receptor within the extracellular N-terminal exodomain. The activating cleavage site is the residue 41–42 (R↓SFLLRN), 36–37(R↓SLIGKV), 38–39 (K↓TFRGAP) and 47–48 (R↓GYPGQV) for human PAR1, PAR2, PAR3 and PAR4, respectively . The cleavage unmasks a new N-terminus, which, in turn, acts as a tethered ligand sequence. The tethered ligand domain binds intramolecularly to and activates the receptor, thus initiating signaling. The proteases that activate PARs include coagulation factors (e.g. thrombin, coagulation factors VIIa and Xa), proteases from inflammatory cells (e.g., mast cell tryptase, neutrophil cathepsin G) and enzymes from epithelial tissues (e.g., trypsins). PAR1, PAR3 and PAR4 are activated principally by thrombin, while PAR2 is activated by trypsin-like proteases such as mast cell tryptase and coagulation Factor Xa. Synthetic PAR-activating peptides (PAR-APs), corresponding to the first five or six amino acids of the tethered ligand sequences, can directly activate PARs, except for PAR3 [3, 9–13]. Since these synthetic peptides function as receptor agonists independent of proteolysis, PAR-APs are useful for studying the physiological and pathophysiological functions of PARs.
PARs are found in a large variety of normal and malignant tissues and cells including skin, platelets, endothelial cells, gastrointestinal tract, brain and lungs. Most cell types express several PARs; for example, A431 cells endogenously express PAR1 and PAR2 [14, 15]. The presence of several PARs in a cell type makes it unclear how the cell differentiates among these signaling. Recently, we had developed a non-invasive and manipulation-free cell assay technology, termed MRCAT (Mass Redistribution Cell Assay Technology), which is centred on resonant waveguide grating (RWG) biosensor . The RWG biosensor directly measures ligand-induced dynamic mass redistribution (DMR) within the bottom portion of adherent cells. Theoretical and numerical analysis suggests that the resultant DMR signal such as the Gq-type DMR  represents a novel physiological readout for cell signaling, which consists of contributions of many cellular events downstream the ligand-induced receptor activation. The MRCAT enables the study of systems cell biology of receptors including epidermal growth factor receptor (EGFR)  and bradykinin B2 receptor . Here we applied the MRCAT to investigate the actions of several PAR agonists, with a special emphasis on the roles of cholesterol in regulating the signaling of endogenous PARs in A431 cells.
PAR1 and PAR2 transduce Gq/11 signaling in A431
EC50 values of PAR agonists in A431. EC50 values were obtained using conventional Ca2+ flux assay, in comparison with those obtained using the MRCAT. In the case of MRCAT data, the amplitudes of both P-DMR and N-DMR events as a function of agonist concentration (as indicated in Fig. 1c) were used to calculate EC50.
EC50(n = 3)
45.7 ± 5.8 nM
98.0 ± 27.8 nM
102.1 ± 21.9 nM
2.5 ± 0.3 μM
2.3 ± 0.6 μM
3.2 ± 0.8 μM
3.8 ± 0.4 μM
6.1 ± 1.0 μM
9.1 ± 2.2 μM
6.0 ± 1.0 unit/ml
9.6 ± 2.0 unit/ml
11.0 ± 1.9 unit/ml
5.0 ± 0.4 μM
1.9 ± 0.1 μM
3.1 ± 0.2 μM
Functional interactions between PAR1 and PAR2
Since A431 expresses both PAR1 and PAR2, we were interested in the possibility of functional interactions between the two receptors. First, we examined the maximal responses induced by PAR agonists (Fig. 2). The maximal [Ca2+]i elevation induced by trypsin, SLIGRL-amide, SLIGKV-amide, thrombin, and SFLLR-amide was found to be 100 ± 6%, 64 ± 4%, 52 ± 6%, 48 ± 4%, and 74 ± 4%, respectively. The maximal [Ca2+]i elevation induced by trypsin was approximately 2 fold as high as those induced by thrombin, SLIGKV-amide or SLIGRL-amide, whereas SFLLR-amide at 80 μM led to an intermediate maximal [Ca2+]i elevation. Furthermore, a mixture of SFLLR-amide and SLIGKV-amide (both at 20 μM) resulted in an [Ca2+]i elevation of 102 ± 5.4%. At saturating concentrations, these agonists also led to similar DMR signals but with different maximal amplitudes (Fig. 2a). The maximal amplitudes of these DMR signals had an order that is almost identical, but less pronounced, to those obtained using Ca2+ flux measurements (Fig. 2b). It is worthy noting that the PAR2-specific agonist SLIGKV-amide led to relatively lower maximal responses, in terms of both Ca2+ mobilization and DMR signal, than another PAR2-specific agonist SLIGRL-amide did.
Differentiated regulation of PAR1 and PAR2 signaling
There is growing evidence that GPCR signaling is complicated – many GPCRs including PAR2 elicit both G protein-dependent and independent signaling events [20, 31]. To complicate this, a GPCR may exist in a collection of microstates (i.e., conformations), and different agonists may result in distinct active-state conformations, thus directing the receptor-induced signals to various cellular pathways [32, 33]. Conventional cell-based assays typically measure a singular cellular response (e.g., second messenger generation, protein interactions or trafficking). Because of that, these assays may lead to false negatives, due to alternative pathway a ligand may selectively activate. On the other hand, the non-invasive optical biosensor used here utilizes an evanescent wave with a short penetration depth (~200 nm) to probe ligand-induced dynamic redistribution of cellular matter; the resultant DMR signal is an integrated cellular response . Thus, the use of the DMR signal as an alternative readout for receptor activation is advantageous. Since many cell signaling events involve significant redistribution of cellular matters, the biosensor-based assays may find broad applicability in many different types of targets including GPCRs [16, 18, 34], EGFR  and ion channels (unpublished data). In A431 cells, we recently have identified three classes of DMR signals induced by panels of GPCR agonists targeting several endogenous receptors, each of which was correlated well with the activation of a class of GPCRs, depending on the G protein with which the receptor is coupled (i.e., Gq, Gs and Gi).
Because of the short penetration depth of the evanescent wave of the present biosensor, only cellular events occurring within the detection zone of the cell layer contribute to ligand-induced DMR signals. Although ligand-induced receptor activation may lead to an array of signaling pathways or events [16–18], only signaling events having significant redistribution of cellular matters could be detected. Recently we have developed a mathematical model for the DMR signals mediated by Gq-coupled receptors . We also have shown that a GPCR ligand-induced DMR signal primarily consists of three components: trafficking of intracellular targets to the activated receptors and subsequently receptor internalization , changes in cell adhesion degree , and cytoskeletal remodelling which is at the crossroad of the receptor activation to downstream signaling events [, unpublished data].
Here we examined the signaling of endogenous PARs in A431. Among PAR agonists examined, only PAR1 and PAR2 agonists resulted in significant Ca2+ mobilization and Gq-type DMR signals. This result is consistent with the fact that only PAR1 and PAR2 are endogenously expressed in A431, and both receptors elicit Gq signaling. The DMR signals induced by all PAR1 and PAR2 agonists share almost identical dynamics, except of the signaling amplitudes (Fig. 2a). The overall dynamics – an initial rapid P-DMR event followed by a relatively slow N-DMR event – is also similar to those mediated through the activation of other endogenous Gq-coupled receptors in A431 cells [[17, 34]; unpublished data]. Our recent theoretical analysis suggests that for Gq-coupled receptors, the P-DMR is primarily resulted from the recruitment of intracellular targets to the activated receptors at the cell membrane, while the receptor internalization is a major contributor to the N-DMR event . Furthermore, the PAR agonist-induced DMR signals also share similarity with the Ca2+ ionophore A23187-induced DMR signal (Fig. 7c), suggesting that the DMR signals of PAR agonists obtained are mainly downstream of Ca2+ pathway. In addition, compared to Ca2+ mobilization signals, the less pronounced difference in the maximal DMR responses of different PAR agonists also indicates that the DMR signals are downstream of Ca2+ mobilization. It is a recent finding that in many cases an agonist-induced maximal response, a measure of agonist efficacy, is dependent on the cellular events measured. This is because GPCR signaling typically proceeds through a series of amplification steps. As a result, the closer to the receptor activation step the cellular event measured is, the bigger difference the agonist efficacy might be . Together, these results suggest that the DMR signals of PAR agonists are primarily resulted from Gq signaling, although one cannot rule out the contributions of other signaling pathways to the overall DMR signal.
Nonetheless, similar to Ca2+ mobilization, the ligand-induced DMR signals are not only dependent on and saturable to ligand concentrations (Fig. 1), but also show classical desensitization patterns upon repeated agonist stimulation (Fig. 5), suggesting that the DMR signal can serve as a novel readout for monitoring receptor activation. Interestingly, the two PAR2-specific agonists SLIGRL-amide and SLIGKV-amide-induced maximal responses, measured using both Ca2+ flux and MRCAT assays, were significantly different (Fig. 2b). Such difference may be due to the functional selectivity of G protein signaling by soluble PAR agonists .
Three lines of evidences suggest that there are functional interactions between PAR1 and PAR2 in A431. First, among PAR1,2 agonists examined, trypsin resulted in the highest Ca2+ mobilization, while SFLLR-amide led to an intermediate Ca2+ mobilization. Similar trend was also observed in their DMR signals (Fig. 2). Co-stimulation with SFLLR-amide and SLIGKV led to Ca2+ mobilization or a DMR signal that is at the level similar to that induced by trypsin alone. These results suggest that: (i) the soluble PAR1 ligand SFLLR-amide may partially activate PAR2, and (ii) trypsin may transactivate PAR1 through unknown mechanism(s). Secondly, the desensitization patterns, as examined using repeated stimulation with various combinations of PAR agonists, also support the functional interactions between PAR1 and PAR2. The trypsin-treated cells lost their responsiveness to either PAR agonist examined, but not to bradykinin, while the thrombin-treated cells still respond to trypsin. Thirdly, a PAR1 partial agonist YFLLRNP-amide can attenuate the DMR signals induced by thrombin or SFLLR-amide, but not SLIGKV-amide. At 729 μM YFLLRNP-amide totally blocked the DMR signal induced by thrombin, but only partially attenuated those induced by either SFLLR-amide or trypsin. Collectively, these results suggest that both SFLLR-amide and trypsin might activate both receptors.
Although it appears that both receptors elicit Gq signaling in A431, there is distinct difference in the kinetics of receptor re-sensitization. The prolonged stimulation (~1 hr) with trypsin resulted in complete desensitization of cells to sequential stimulation with thrombin, but partial desensitization to SFLLR-amide, SLIGKV-amide or SLIGRL-amide (Fig. 5). On the other hand, the cells still respond to trypsin, after pre-stimulation with thrombin, SFLLR-amide, SLIGKV-amide, or SLIGRL-amide. These suggest that PAR2 resensitizes much faster than PAR1. It is known that receptor proteolysis and phosphorylation regulate the activities of PARs through receptor internalization and the inhibition of intracellular signal transduction [9, 36, 37]. Depending on the cellular context, the recovery of functional receptors at the cell surface could take from tens of minutes to hours [38, 39].
The almost identical sensitivity of both trypsin- and thrombin-induced Ca2+ mobilization to cholesterol removal suggests that the cell surface cholesterol level plays an equally important role in regulating the amplitudes of Ca2+ mobilization induced by the activation of both PAR1 and PAR2. It is known that cholesterol extraction leads to the loss of compartmentalization of PtdIns 4,5-P2, and Gq, two important molecules for PAR signaling [40, 41]. The suppression of Ca2+mobilization by cholesterol depletion might be a direct result of delocalization of PtdIns and Gq.
Interestingly, the DMR signals induced by trypsin or thrombin exhibited different dependency on the concentration of mβCD in solution. The pre-treatment of cells with mβCD but not its inactive analog αCD attenuated PAR signaling including Ca2+ mobilization and DMR signals induced by thrombin or trypsin. The partial inhibitory effect of EGF pre-treatment suggests that the transactivation of EGFR by cholesterol depletion may attenuate, directly or indirectly, the N-DMR event mediated by thrombin or trypsin (Fig. 8b and 8c). Since the DMR signal is an integrated cellular response, it is very sensitive to the cellular background [17, 18]. The activation of EGFR directly by EGF, or indirectly by cholesterol depletion, could alter the cellular background, thus indirectly impairing the N-DMR event induced by both PAR agonists. Alternatively, the EGFR activation or transactivation could lead to signaling pathway(s) crosstalking with GPCR signaling.
For PAR1, the cell surface cholesterol level seems equally regulate both Ca2+ mobilization and DMR signal, because both types of cellular responses induced by thrombin exhibited the same sensitivity to mβCD concentration in solution. Conversely, for PAR2 the cell surface cholesterol level appears regulate Ca2+ mobilization and DMR signal differently. Such a difference in sensitivity to mβCD concentration between the trypsin- and thrombin-induced DMR signals suggest that two receptors may involve different cellular mechanism(s) or signaling network interactions.
The signaling of endogenous PAR1 and PAR2 in A431 was studied using non-invasive and manipulation-free optical biosensor. The biosensor-manifested DMR signals follow classical receptor biology. Similar to Ca2+ mobilization, the DMR signals are saturable to ligand concentrations; exhibit comparable desensitization patterns in response to repeated stimulation with various combinations of agonists; and are sensitive to cell surface cholesterol level. More significantly, data analysis suggests that the biosensor differentiates the signaling of PAR1 and PAR2 in A431 under physiologically relevant conditions.
Thrombin, trypsin, methyl-β-cyclodextrin (mβCD), A23187, AG1478, α-cyclodextrin (αCD), and epidermal growth factor (EGF) were purchased from Sigma Chemical Co. (St. Louis, MO). Fluo-3 was obtained from Molecular Probes (Eugene, OR). SFLLR-amide, SLIGKV-amide, SLIGRL-amide, bradykinin, TFRGAP, GYPGQV, and YFLLRNP-amide were obtained from Bachem (King of Prussia, PA). All compounds were used as received. Corning® Epic™ 96well biosensor microplates were obtained from Corning Inc (Corning, NY), and cleaned by exposure to high intensity UV light (UVO-cleaner, Jelight Company Inc., Laguna Hills, CA) for 6 minutes before use.
Human epidermoid carcinoma A431 cells (American Type Cell Culture) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 4.5 g/liter glucose, 2 mM glutamine, and antibiotics. ~5 × 104 cells at passage 3 to 8 suspended in 200 μl the DMEM medium containing 10% FBS were placed in each well of a 96well microplate, and were cultured at 37°C under air/5% CO2 for ~2 days, followed by ~20 hr starvation through continuously culturing in the serum-free DMEM.
Fluo-3 Ca2+ mobilization assay
Cells were grown in Costar™ 96well clear cell culture microplates. After starvation, the cells were washed with 1× HBSS (1× regular Hank's balanced salt solution, 20 mM HEPES buffer, pH 7.0) in the presence of 2.5 mM probenicid, and labeled in the same buffer containing 4 μM Fluo-3 for 1 hour at room temperature. The cells were then washed twice with buffer, maintained with 100 μl 1× HBSS containing 2.5 mM probenicid. The assay was initiated by transferring 50 μl PAR agonist solution to the cell plate, and calcium signal was recorded over 6 minutes with a 6 sec interval using HTS7000 BioAssay Reader (PerkinElmer Life Science, Boston, MA). The fluorescent intensity before stimulation was recorded and used as a baseline. The percentage increase in fluorescence intensity after stimulation, relative to the baseline fluorescence, was analyzed and used directly as a measure for the increase of intracellular Ca2+ level induced by PAR agonists.
Optical biosensor measurements
Corning® Epic™ angular interrogation system with transverse magnetic or p-polarized TM0 mode was used for all studies. The detailed instrumental setup and assay protocols had been previously described [16–18]. Briefly, all compound solutions were prepared using 1 × HBSS containing minimal amount of dimethyl sulfoxide, while the starved cells were washed and maintained with 100 μl the serum-free DMEM. The cells were then treated with 50 μl 1 × HBSS buffered solution in the absence and presence of a compound, followed by stimulation with ligand solutions. The cellular responses were monitored in real time throughout the assays.
For functional recovery after cholesterol removal with mβCD, the quiescent A431 cells were treated with 5 mM mβCD for 15 minutes to ensure the removal of cell surface cholesterol content, followed by washing the treated cells three times with the medium only. The cells were then maintained with 100 μl the medium, and placed into the optical systems. After incubation for 15 minutes to allow cells reaching reasonably steady state, a 100 μl solution of thrombin at 80 unit/ml was added to each well at specific time. The optical responses were recorded throughout the assays.
Unless specifically mentioned, three replicates were carried out for each measurement or each compound. The standard deviation was derived from these measurements (n = 3). The assay coefficient of variation was found to be typically less than 10%. All dose-dependent responses were analyzed using non-linear regression method with the Prism software (Graph Pad).
The authors would like to thank Dr. Norman H. Fontaine for his support in instrumentation.
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