Optical biosensor differentiates signaling of endogenous PAR₁and PAR₂in A431 cells

Abstract

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.