Optical biosensor differentiates signaling of endogenous PAR1 and PAR2 in A431 cells

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

The Ca²⁺ mobilization and DMR signals mediated through endogenous PARs in A431. (a) The endogenous PARs and their corresponding agonists. Both receptors mediate G_q signaling, which proceeds through activation of the receptor, its coupled G protein and downstream target phospholipase C (PLC). The PLC hydrolyzes the membrane lipid phosphatidylinositol bisphosphate (PIP₂), producing inositol triphosphate (IP₃) and diacylglycerol (DAG). IP₃ binds to and opens a calcium channel in the endoplasmic reticulum, leading to calcium mobilization. Calcium alters many cellular processes. The interaction of both DAG and calcium with protein kinase C (PKC) activates PKC kinase activity, which, in turn, phosphorylates many different protein targets including small GTPase Rho, leading to the remodeling of cytoskeletal structure. (b) The increase in intracellular Ca²⁺ level as a function of the concentration of different soluble PAR agonists. (c) The real-time dynamic mass redistribution signals induced by SFLLR-amide at different doses. The solid arrow indicates the time when SFLLR-amide is introduced. The DMR consists of two phases: an increase signal (termed Positive-DMR, P-DMR) and a sequential decay signal (termed Negative-DMR, N-DMR). (d) The amplitudes of both P-DMR and N-DMR events, calculated as indicated in (c), as a function of SFLLR-amide concentration.

Figure 2

Correlation between the maximal Ca²⁺ mobilization and DMR responses induced by PAR agonists. (a) The DMR signals induced by PAR agonists: TFRGAP (20 μM), GYPGQV (20 μM), SLIGRL-amide (20 μM), SLIGKV-amide (20 μM), thrombin (40 unit/ml), SFLLR-amide (20 μM), trypsin (1024 nM), SFLLR-amide+SLIGRL-amide (each at 20 μM). (b) Comparison of the maximal DMR and Ca²⁺ mobilization responses induced by different PAR agonists. The DMR response was calculated using the amplitude of the P-DMR event. Since trypsin at doses greater than ~1000 nM led to significant cell detachment (ref. 16), the DMR signal induced by trypsin at 1024 nM was used as its maximal response.

Figure 3

The effect of YFLLRNP-amide on the DMR signals induced by PAR agonists. The cells were pre-treated with YFLLRNP-amide at different doses. The amplitudes of the P-DMR events induced by each agonist (40 unit/ml thrombin, 20 μM SFLLR-amide, or 20 μM SLIGKV-amide) were plotted as a function of YFLLRNP-amide concentration.

Figure 4

Desensitization of A431 cells to repeated agonist stimulation – Ca²⁺ mobilization. The cells were subject to repeated stimulation, separated by 6 min, with various combinations of agonists. The agonist concentration was 40 unit/ml, 200 nM, 20 μM, 20 μM, and 100 nM for thrombin, trypsin, SFLLR-amide, SLIGKV-amide, and bradykinin, respectively.

Figure 5

Desensitization of A431 cells to repeated agonist stimulation – DMR signal. The cells were subject to repeated stimulation, separated by ~1 hr, with various combinations of agonists. The agonist concentration was 40 unit/ml, 200 nM, 20 μM, 20 μM, and 100 nM for thrombin, trypsin, SFLLR-amide, SLIGKV-amide, and bradykinin, respectively.

Figure 6

Cholesterol removal impairs the agonist-induced Ca²⁺ mobilization. mβCD was used to deplete cell surface cholesterol. Its effect on agonist-induced Ca²⁺ mobilization was examined. The agonists were trypsin (200 nM) and thrombin (40 unit/ml).

Figure 7

Cholesterol removal attenuates the agonist-induced DMR signals. mβCD was used to deplete cell surface cholesterol. Its effect on the amplitudes of the P-DMR events induced by (a) thrombin (40 unit/ml) or (b) trypsin (200 nM) was analyzed. In comparison, the effect of αCD was also included. (c) The DMR signals of A431 cells induced by A23187 without or with the pre-treatment with 5 mM mβCD.

Figure 8

The functional recovery of PAR signaling after cholesterol removal. mβCD was used to extract cell surface cholesterol. After the mβCD-containing medium was replaced with the DMEM, thrombin was introduced to stimulate cells at different time. Each graph is an average of 7 independent responses.

Figure 9

The effect of preceding EGF stimulation on PAR signaling. (a) The Ca²⁺ mobilization. (b, c) The DMR signals. The ligands were EGF (100 nM), trypsin (100 nM), and thrombin (40 unit/ml). The cell responses with the pre-treatment with the HBSS only were also included as control.