Differential Roles of Arrestin-2 Interaction with Clathrin and Adaptor Protein 2 in G Protein-coupled Receptor Trafficking

The non-visual arrestins, arrestin-2 and arrestin-3, play a critical role in regulating the signaling and trafficking of many G protein-coupled receptors (GPCRs). Molecular insight into the role of arrestins in GPCR trafficking has suggested that arrestin interaction with clathrin, β 2 -adaptin (the β-subunit of the adaptor protein AP2), and phosphoinositides contributes to this process. In the present study, we have attempted to better define the molecular basis and functional role of arrestin-2 interaction with clathrin and β 2 -adaptin. Site-directed mutagenesis revealed that the C-terminal region of arrestin-2 mediated β 2 -adaptin and clathrin interaction with Phe-391 and Arg-395 having an essential role in β 2 -adaptin binding and LIELD (residues 376–380) having an essential role in clathrin binding. Interestingly, arrestin-2-R169E, an activated form of arrestin that binds to GPCRs in a phosphorylation-independent manner, has significantly enhanced binding to β 2 -adaptin and clathrin. This suggests that receptor-induced conformational changes in the C-terminal tail of arrestin-2 will likely play a major role in mediating arrestin interaction with clathrin-coated pits. In an effort to clarify the role of these interactions in GPCR trafficking we generated arrestin mutants that were completely and selectively defective in either clathrin (arrestin-2-ΔLIELD) or β 2 -adaptin (arrestin-2-F391A) interaction. Analysis of these mutants in COS-1 cells revealed that arrestin/clathrin interaction was essential for agonist-promoted internalization of the β 2 -adrenergic receptor, while arrestin/β 2 -adaptin interaction appeared less critical. Arrestin-2 mutants defective in both clathrin and β 2 -adaptin binding functioned as effective dominant negatives in HEK293 cells and significantly attenuated β 2 -adrenergic receptor internalization. These mutants should prove useful in better defining the role of arrestins in mediating receptor trafficking.