Palmitoylation of human proteinase-activated receptor-2 differentially regulates receptor-triggered ERK1/2 activation, calcium signalling and endocytosis

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 G_i, 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.