Palmitoylation of PAR1 regulates the accessibility of C‐tail YXX φ motifs to AP‐2 and internalization

Abstract

Protease‐activated receptor‐1 (PAR1), a G‐protein coupled receptor (GPCR) for thrombin, is irreversibly and proteolytically activated. Thus, desensitization and intracellular trafficking of PAR1 are critical for proper cellular responses. Palmitoylation of GPCRs regulates various aspects of signaling and trafficking, however the function of palmitoylation in PAR1 signal regulation is not known. We examined palmitoylation in PAR1 wildtype and in a mutant where C‐tail cysteines were converted to alanines. Here, we show that PAR1 is palmitoylated at these C‐tail cysteine residues. Moreover, the PAR1 palmitoylation mutant displayed enhanced constitutive internalization, whereas agonist‐induced internalization was comparable to wildtype receptor. Constitutive internalization of the PAR1 palmitoylation mutant was dependent on the clathrin adaptor protein complex AP‐2, like wildtype receptor. However, unlike wildtype PAR1, AP‐2‐mediated internalization required the proximal rather than the distal YXXφ motif in this mutant. The PAR1 palmitoylation mutant also displayed defects in phosphorylation and ubiquitination. Thus, palmitoylation stabilizes a conformation of the PAR1 C‐tail that is important for trafficking and other post‐translational modifications. Supported by RO1 HL073328 & UC TRDRP.