title: The role of palmitoylation in signalling, cellular trafficking and plasma membrane localization of protease-activated receptor-2.
abstract: Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. This irreversible activation mechanism leads to rapid receptor desensitization by internalisation and degradation. We have explored the role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Experiments using the palmitoylation inhibitor 2-bromopalmitate indicated that palmitate addition is important in trafficking of PAR2 endogenously expressed by prostate cancer cell lines. This was supported by palmitate labelling using two approaches, which showed that PAR2 stably expressed by CHO-K1 cells is palmitoylated and that palmitoylation occurs on cysteine 361. Palmitoylation is required for optimal PAR2 signalling as Ca²⁺ flux assays indicated that in response to trypsin agonism, palmitoylation deficient PAR2 is ∼9 fold less potent than wildtype receptor with a reduction of about 33% in the maximum signal induced via the mutant receptor. Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. We also show that receptor palmitoylation occurs within the Golgi apparatus and is required for efficient agonist-induced rab11a-mediated trafficking of PAR2 to the cell surface. Palmitoylation is also required for receptor desensitization, as agonist-induced β-arrestin recruitment and receptor endocytosis and degradation were markedly reduced in CHO-PAR2-C361A cells compared with CHO-PAR2 cells. These data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor.
authors.affiliation: Mater Medical Research Institute, Aubigny Place, Raymond Terrace, South Brisbane, Queensland, Australia.
authors.forename: Mark N
authors.initials: MN
authors.lastname: Adams
authors.forename: Melinda E
authors.initials: ME
authors.lastname: Christensen
authors.forename: Yaowu
authors.initials: Y
authors.lastname: He
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Waterhouse
authors.forename: John D
authors.initials: JD
authors.lastname: Hooper
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000378:metabolism; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000378:metabolism; D004705:Endocytosis; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D054878:Lipoylation; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010168:Palmitates / Q000378:metabolism; D021381:Protein Transport; D059748:Proteolysis; D044464:Receptor, PAR-2 / Q000819:agonists / Q000737:chemistry / Q000378:metabolism*; D055571:Secretory Pathway; D015398:Signal Transduction; D000071557:beta-Arrestins; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
chemical_list: D019390:Arrestins; D003545:Cysteine; D010168:Palmitates; D044464:Receptor, PAR-2; D000071557:beta-Arrestins; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins