Palmitoylation of protease‐activated receptor‐1: regulation of receptor ubiquitination and intracellular trafficking

Protease‐activated receptor‐1 (PAR1), a G‐protein coupled receptor (GPCR) for thrombin, is irreversibly proteolytically activated. Thus, desensitization and intracellular trafficking are critical for regulating the fidelity of PAR1 signaling. Palmitoylation of GPCRs has diverse functions in the regulation of receptor signaling and trafficking, yet the function of palmitoylation in PAR1 signal regulation is not known. To delineate the role of palmitoylation, we examined the palmitoylation status of PAR1 wildtype and a mutant with C‐tail cysteines (C 387 and C 388 ) converted to alanines (A). Here, we show that PAR1 is basally palmitoylated at C 387 and C 388 residues. Moreover, the PAR1 CC/AA palmitoylation‐deficient mutant displayed enhanced constitutive internalization compared to wildtype receptor, whereas agonist‐induced PAR1 internalization remained intact. Constitutive internalization of PAR1 CC/AA was dependent on AP‐2, like wildtype receptor. Interestingly, a substantial amount of PAR1 CC/AA mutant was retained within the trans ‐Golgi and showed increased ubiquitination, whereas activation of surface localized PAR1 CC/AA mutant failed to undergo ubiquitination. Thus, palmitoylation is likely to stabilize the proper PAR1 C‐tail conformation important for regulation of receptor signaling and trafficking. Supported by NIGMS T32 Pharmacological Sciences Training and TRDRP Grants.