Fig. 11.
Selective activation of p42/44 MAP kinase in PAR2-transfected KNRK cells by SLAAAA-NH2 compared with the TL mutant-derived synthetic peptides AAIGRL-NH2 and LSIGRL-NH2. KNRK cell lines expressing wt-rPAR2, rPAR2-Leu37Ser38, and rPAR2-Ala37–38 were treated for 10 min at 37°C with the synthetic PAR2 tethered ligand-related peptides, SLAAAA-NH2, LSIGRL-NH2, and AAIGRL-NH2 or with trypsin, and the activation of MAP kinase was monitored (P-p42/44 relative to T-p42/44) by Western blot analysis as outlined by the legend to Fig. 2. A, representative Western blot detection of the activation of MAP kinase (P-42/44, relative to T-p42/44) in wt-rPAR2 by SLAAAA-NH2 (third lane from left) but not by LSIGRL-NH2 and AAIGRL-NH2 (second and fourth lanes from left). B, representative western blots for the activation of MAP kinase (P-p42/44, relative to T-p42/44) in wt-rPAR2 (top), rPAR2-Leu37Ser38 (middle), and rPAR2-Ala37–38 (bottom) by trypsin (10 nM), SLIGRL-NH2 (10 μM), or SLAAAA-NH2 (10 μM). C, quantitative densitometric image analysis for the percentage increase over baseline (no treatment, NT) of P-p42/44, normalized to T-p42/44, as shown in B, for activation of wt-rPAR2 (solid histograms), rPAR2-Leu37Ser38 (open histograms), and rPAR2-Ala37–38 (gray histograms). The quantitative densitometry values (histograms) are expressed as averages (± S.E.M., bars) for three or more independently conducted experiments.