Fig 5. A signal peptide is important for functional expression of PAR2.

A. schematic diagram showing the modifications to PAR2 receptor. The N-terminal extracellular sequences of various PAR2 mutants are shown. Human PAR2 wild type (PAR2), PAR2 with the signal peptide deleted (PAR2ΔSP), PAR2 with an insulin signal peptide (PAR2-INSP) and an insulin receptor signal peptide (PAR2-IRSP). The native signal peptide of PAR2 is shown in blue. The insulin and insulin signal peptides are shown in green and purple respectively. The tether ligand sequence of PAR2 (SLIGKV) is highlighted in yellow. B, C. Characterization of PAR2 mutants in FLIPR assay using trypsin or the synthetic PAR2 agonist peptide (PAR2-AP) as the ligands. Expression constructs for PAR2 wild type receptor and various modifications were cloned into pcDNA3.1 and transiently expressed in HEK293 cells with par1 and par2 knocked-out. Various concentrations of trypsin (B) or PAR-AP (C) were added to stimulate the intracellular Ca²⁺ mobilization. Relative fluorescent intensity units (RFU) are shown. The experiments were performed in triplicates at each data point and the results shown are mean ± sd. HEK293 cells with par1 and par2 genes knocked-out were used as the host cells for recombinant expression of various PAR2 receptors. Untransfected cells were used as the negative controls (NC). The experiments have been performed 3 times and very similar results have been observed. Example data is shown.