Ubiquitination differentially regulates clathrin-dependent internalization of protease-activated receptor-1

Abstract

Protease-activated receptor-1 (PAR1), a G protein–coupled receptor (GPCR) for thrombin, is irreversibly activated by proteolysis. Consequently, PAR1 trafficking is critical for the fidelity of thrombin signaling. PAR1 displays constitutive and agonist-induced internalization, which are clathrin and dynamin dependent but are independent of arrestins. The clathrin adaptor AP2 (adaptor protein complex-2) is critical for constitutive but not for activated PAR1 internalization. In this study, we show that ubiquitination negatively regulates PAR1 constitutive internalization and specifies a distinct clathrin adaptor requirement for activated receptor internalization. PAR1 is basally ubiquitinated and deubiquitinated after activation. A PAR1 lysineless mutant signaled normally but was not ubiquitinated. Constitutive internalization of ubiquitin (Ub)-deficient PAR1 was markedly increased and inhibited by the fusion of Ub to the cytoplasmic tail. Ub-deficient PAR1 constitutive internalization was AP2 dependent like the wild-type receptor. However, unlike wild-type PAR1, AP2 was required for the internalization of activated Ub-deficient receptor, suggesting that the internalization of ubiquitinated PAR1 requires different endocytic machinery. These studies reveal a novel function for ubiquitination in the regulation of GPCR internalization.

FBL2 Regulates Amyloid Precursor Protein (APP) Metabolism by Promoting Ubiquitination-Dependent APP Degradation and Inhibition of APP Endocytosis

“…In contrast, our results show that FBL2-mediated ubiquitination of APP inhibits its endocytosis, which is further supported by the inability of the FBL2 deletion mutant lacking ubiquitin ligase activity to inhibit endocytosis and inhibition of APP endocytosis in APP-K651A mutant. This situation is similar to previous reports that ubiquitination negatively regulated internalization of protease-activated receptor-1, a G-protein-coupled receptor for thrombin (Wolfe et al, 2007). This evidence suggests that ubiquitination can both positively and negatively regulate receptor internalization.…”

Transactivation of the PAR1-PAR2 Heterodimer by Thrombin Elicits β-Arrestin-mediated Endosomal Signaling

“…To further examine the possibility that PAR1 directs constitutive trafficking of the heterodimer, we utilized the PAR1 AKKAA mutant, which is defective in AP-2 (adaptor protein complex-2) binding and constitutive internalization (18), and the ubiquitination-deficient PAR1 0K mutant, which exhibits enhanced constitutive internalization mediated by AP-2 (27). Cells were prelabeled with receptor-specific antibodies, washed, and incubated for either 0 or 30 min at 37°C.…”

Regulation of Protease-activated Receptor 1 Signaling by the Adaptor Protein Complex 2 and R4 Subfamily of Regulator of G Protein Signaling Proteins

“…Several other GPCRs have also been shown to internalize independently of ␤-arrestins (8). We showed previously that the clathrin adaptor protein complex 2 (AP-2) and epsin-1 are * This work was supported, in whole or in part, by National Institutes of Health essential for agonist-induced PAR1 internalization (9,10). The clathrin adaptor AP-2 is a heterotetrameric complex comprised of ␣, ␤2, 2, and 2 adaptin subunits and has critical functions in the assembly and recruitment of cargo to clathrin-coated pits.…”