Optical biosensor differentiates signaling of endogenous PAR ₁ and PAR ₂ 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 (PAR ₁) and 2 (PAR ₂) 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 PAR ₁ and PAR ₂ agonists, but neither PAR ₃ nor PAR ₄ agonists, trigger dose-dependent Ca ²⁺ mobilization as well as G _q-type DMR signals. Both Ca ²⁺ flux and DMR signals display comparable desensitization patterns upon repeated stimulation with different combinations of agonists. However, PAR ₁ and PAR ₂ exhibit distinct kinetics of receptor re-sensitization. Furthermore, both trypsin- and thrombin-induced Ca ²⁺ 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 PAR ₁ and PAR ₂ in A431.