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.

Supplemental material for this article may be found at http://mcb.asm.org/.

We thank T. K. Harden and R. A. Nicholas for comments and helpful discussions and David J. Owen and Robert J. Lefkowitz for generously providing reagents.

This work was supported by National Institutes of Health grants HL067697 and HL073328 (to J.T.) and GM065533 (to D.P.S.). M.M.P. and C.A.J. were each supported by American Heart Association Predoctoral Fellowships.