Clathrin Adaptor AP2 Regulates Thrombin Receptor Constitutive Internalization and Endothelial Cell Resensitization

Abstract

Protease-activated receptor 1 (PAR1), a G protein-coupled receptor for the coagulant protease thrombin, is irreversibly activated by proteolysis. Unactivated PAR1 cycles constitutively between the plasma membrane and intracellular stores, thereby providing a protected receptor pool that replenishes the cell surface after thrombin exposure and leads to rapid resensitization to thrombin signaling independent of de novo receptor synthesis. Here, we show that AP2, a clathrin adaptor, binds directly to a tyrosine-based motif in the cytoplasmic tail of PAR1 and is essential for constitutive receptor internalization and cellular recovery of thrombin signaling. Expression of a PAR1 tyrosine mutant or depletion of AP2 by RNA interference leads to significant inhibition of PAR1 constitutive internalization, loss of intracellular uncleaved PAR1, and failure of endothelial cells and other cell types to regain thrombin responsiveness. Our findings establish a novel role for AP2 in direct regulation of PAR1 trafficking, a process critically important to the temporal and spatial aspects of thrombin signaling.

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

“…2A), as the majority of the receptor localized to endosomes after agonist exposure. Thus, PAR1 displays constitutive and agonist-induced internalization in HeLa cells, as we reported previously (18,26). In contrast, incubation with neither thrombin nor medium alone for 30 min at 37°C was sufficient to drive HA-PAR2 internalization (Fig.…”

“…Mouse anti-PAR1 antibody WEDE was from Beckman Coulter (Fullerton, CA). Rabbit anti-PAR1 polyclonal antibody C5433 was described previously (18), and anti-PAR2 polyclonal antibody was from Dr. Wolfram Ruf (The Scripps Research Institute). Rabbit anti-␤-arrestin polyclonal antibody was provided by Dr. Jeffrey Benovic (Thomas Jefferson University).…”

“…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.…”

Activated protein C promotes protease-activated receptor-1 cytoprotective signaling through β-arrestin and dishevelled-2 scaffolds

“…Rac1, caveolin-1, and EEA-1 antibodies were from BD Biosciences. The monoclonal anti-PAR1 WEDE-15 antibody was from Beckman Coulter, and the polyclonal anti-PAR1 rabbit antibody was generated as previously described (40). TRITC-conjugated phalloidin, anti-FLAG, and anti-actin antibodies were obtained from Sigma.…”

Pathophysiological consequences of receptor mistraffic: Tales from the platelet P2Y 12 receptor

“…This pool of receptor, which recycles in a rab4-dependent manner, can readily recycle back to the cell surface (18). Whether like other platelet GPCRs (54,55) this agonist-independent traffic may provide an internal pool of receptor able to mobilise to the cell surface and support receptor signalling requires further study. In cell lines the Arg122Cys P2Y 12 receptor also internalizes in an agonist-independent manner although at a faster rate than the wild type receptor(31).…”