Proteinase-activated receptors are a novel class of G-protein coupled receptors activated by proteolytic cleavage of the N-terminal domain. The proteinase revealed tethered ligand then binds to and activates the receptor. Recently, we have shown that the post-translational modification, N-linked glycosylation has a profound effect on hPAR₂ receptor function (Compton et al., 2002). Therefore we have investigated the role of another post-translational modification, palmitoylation, in regulating hPAR₂ function.

Employing site directed mutagenesis we have mutated the putative palmitoylation site (C361) within
hPAR ₂ and expressed the mutant receptor (hPAR₂C361A) in the Chinese Hamster Ovary PRO5 cell line. FACS analysis was used to compare cell surface expression of hPAR₂C361A mutant and wild-type hPAR₂ (wt-hPAR₂) receptor expressing cell lines. hPAR₂C361A consistently displayed an ~2-fold greater cell surface expression compared to wt-hPAR₂.

Intracellular Ca²⁺ signalling was then employed to compare the functional activity between wt-hPAR₂ and hPAR₂C361A using cell lines with matched cell surface expression. hPAR₂C361A displayed a striking decrease in agonist sensitivity towards trypsin with EC₅₀ values 4-fold greater than that of wt-hPAR₂ (41 ± 4 nM compared to 11 ± 4 nM, mean ± sem) and 6-fold greater EC₅₀ values for the selective hPAR₂ activating peptide SLIGKV-NH 2 (104 ± 2 nM compared to 16 ± 5 nM). In addition, hPAR₂C361A displayed a decrease of ~50% and ~66% in maximal response to trypsin and SLIGKV-NH₂ respectively, compared to that of wt-PAR₂.

Thus, palmitoylation may play an important role in regulating hPAR₂ function.

Compton, S.J. et al. (2002). Biochem J, 368, 495-505