Fig. 2.

Design and screening of agonist and antagonist PAR2 i3-loop pepducins. (A) Agonist and antagonist activity of third intracellular (i3) loop PAR2 pepducins using calcium flux assays with SW620 colon adenocarcinoma cells that endogenously express PAR2. Each column of three calcium flux traces corresponds to the i3 pepducin sequence shown above. The Top row is the agonist activity of 3–4 μM pepducin and the Middle row is the agonist activity of 14–15 μM pepducin. The Bottom row depicts the calcium signal of 100 μM SLIGRL (open arrowheads) following 1-min pretreatment with 6 μM pepducin (closed arrowheads). Final concentration of DMSO vehicle was 0.2%. (B) Model of the WT PAR2 i3 pepducin P2pal-21 bound to the intracellular surface of PAR2. The location of the i3 pepducin was derived by substituting the coordinates of the i3 loop on the intact receptor with the i3 pepducin using the PAR2 dimer model of Fig. 1A. Key pharmacophores M274 (brown), R284 and K287 (yellow), and palmitate (green) are shown. (C) Agonist activity for each pepducin from A is reported as initial velocity of calcium flux at 3–4 μM pepducin (†), or at 14–15 μM pepducin (‡). Antagonist activity of 6 μM pepducin against 100 μM SLIGRL was measured by area under the curve of calcium flux from the Bottom row of A. Experiments were repeated at least two to three times each and gave highly similar results.