Fig. 1.

Constitutive and ligand-dependent activity of PAR2 is regulated by juxtamembrane residues located in the third intracellular (i3) loop. (A) Location of PAR2 mutants and model of the receptor dimer used in this study. The PAR2 model depicting the i3 loop–eighth helix dimer interface was constructed using X-ray structures of rhodopsin as described in Materials and Methods. (B) Migration of HEK cells transiently transfected with PAR2 (wild type, WT), PAR2-R36A, PAR2-RQ, or PAR2-R36A plus PAR2-RQ toward chemotactic gradients of 30 nM trypsin or DMEM media alone for 18 h in a transwell apparatus (8-μm pore), n = 2, repeated three independent times. (C–E) Constitutive and SLIGRL-activated signaling of PAR2 mutants to PLC-β. PLC-β activity was measured by [³H]-InsP formation over 30 min (39) and was typically stimulated four- to fivefold above basal with 30–100 μM SLIGRL. Mean PAR2 mutant surface expression was assessed by FACS and was 1.0 ± 0.3-fold relative to wild type in COS7 cells and 1.00 ± 0.18 in HEK cells (Fig. S1D). Data (n = 2–8) represent the mean ± SD. *P < 0.05 and ^#P = 0.07.