hPAR2 (human proteinase-activated receptor-2) is a member of the novel family of proteolytically activated GPCRs (G-protein-coupled receptors) termed PARs (proteinase-activated receptors). Previous pharmacological studies have found that activation of hPAR2 by mast cell tryptase can be regulated by receptor N-terminal glycosylation. In order to elucidate other post-translational modifications of hPAR2 that can regulate function, we have explored the functional role of the intracellular cysteine residue Cys361. We have demonstrated, using autoradiography, that Cys361 is the primary palmitoylation site of hPAR2. The hPAR2C361A mutant cell line displayed greater cell-surface expression compared with the wt (wild-type)-hPAR2-expressing cell line. hPAR2C361A also showed a decreased sensitivity and efficacy (intracellular calcium signalling) towards both trypsin and SLIGKV. In stark contrast, hPAR2C361A triggered greater and more prolonged ERK (extracellular-signal-regulated kinase) phosphorylation compared with that of wt-hPAR2 possibly through Gi, since pertussis toxin inhibited the ability of this receptor to activate ERK. Finally, flow cytometry was utilized to assess the rate and extent of receptor internalization following agonist challenge. hPAR2C361A displayed faster internalization kinetics following trypsin activation compared with wt-hPAR2, whereas SLIGKV had a negligible effect on internalization for either receptor. In conclusion, palmitoylation plays an important role in the regulation of PAR2 expression, agonist sensitivity, desensitization and internalization.

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