Adaptor Protein Complex‐2 and epsin‐1 mediate Protease‐activated Receptor‐1 internalization via phosphorylation‐ and ubiquitination‐dependent sorting signals

Abstract

Signaling by protease‐activated receptor‐1 (PAR1), a G protein‐coupled receptor (GPCR) for thrombin, is tightly regulated. PAR1 desensitization is mediated by β‐arrestins. However, internalization of PAR1 occurs through a clathrin‐ and dynamin‐dependent pathway independent of β‐arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex‐2 (AP‐2). However, AP‐2 depletion only partially inhibits agonist‐induced internalization of PAR1, suggesting a function for other clathrin adaptors. We now report that AP‐2 and epsin‐1 are both critical mediators of agonist‐stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin‐interacting motifs of epsin‐1 are required for epsin‐1‐dependent internalization of activated PAR1. AP‐2 regulates activated PAR1 internalization via recognition of distal C‐tail phosphorylation sites rather than the canonical tyrosine‐based motif. We further demonstrate a new function for AP‐2 in modulating the signaling responses of activated PAR1. Thus, AP‐2 and epsin‐1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This work was supported by National Institutes of Health Grant and a University of California Tobacco‐related Disease Research Program Exploratory Award.