Clathrin adaptor AP2 regulates thrombin receptor constitutive internalization and endothelial cell resensitization

Protease-activated receptor I (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 PARI and is essential for constitutive receptor internalization and cellular recovery of thrombin signaling. Expression of a PARI tyrosine mutant or depletion of AP2 by RNA interference leads to significant inhibition of PARI constitutive internalization, loss of intracellular uncleaved PARI, 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 PARI trafficking, a process critically important to the temporal and spatial aspects of thrombin signaling.