β-Arrestin/AP-2 Interaction in G Protein-coupled Receptor Internalization

Abstract

β-Arrestins, proteins involved in the turn-off of G protein-coupled receptor (GPCR) activation, bind to the β2-adaptin subunit of the clathrin adaptor AP-2. The interaction of β2-adaptin with β-arrestin involves critical arginine residues in the C-terminal domain of β-arrestin and plays an important role in initiating clathrin-mediated endocytosis of the β2-adrenergic receptor (β2AR) (Laporte, S. A., Oakley, R. H., Holt, J. A., Barak, L. S., and Caron, M. G. (2000) J. Biol. Chem. 275, 23120-23126). However, the β-arrestin-binding site in β2-adaptin has not been identified, and little is known about the role of β-arrestin/AP-2 interaction in the endocytosis of other GPCRs. Using in vitro binding assays, we have identified two glutamate residues (Glu-849 and Glu-902) in β2-adaptin that are important in β-arrestin binding. These residues are located in the platform subdomain of the C terminus of β2-adaptin, where accessory/adapter endocytic proteins for other classes of receptors interact, distinct from the main site where clathrin interacts. The functional significance of the β-arrestin/AP-2/clathrin complex in the endocytosis of GPCRs such as the β2AR and vasopressin type II receptor was evaluated using mutant constructs of the β2-adaptin C terminus containing either the clathrin and the β-arrestin binding domains or the β-arrestin-binding domain alone. When expressed in human embryonic kidney 293 cells, both constructs acted as dominant negatives inhibiting the agonist-induced internalization of the β2AR and the vasopressin type II receptor. In addition, although the β2-adaptin construct containing both the clathrin and β-arrestin binding domains was able to block the endocytosis of transferrin receptors, a β2-adaptin construct capable of associating with β-arrestin but lacking its high affinity clathrin interaction did not interfere with transferrin receptor endocytosis. These results suggest that the interaction of β-arrestin with β2-adaptin represents a selective endocytic trigger for several members of the GPCR family.