title: Differential roles of arrestin-2 interaction with clathrin and adaptor protein 2 in G protein-coupled receptor trafficking.
abstract: 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, beta(2)-adaptin (the beta-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 beta(2)-adaptin. Site-directed mutagenesis revealed that the C-terminal region of arrestin-2 mediated beta(2)-adaptin and clathrin interaction with Phe-391 and Arg-395 having an essential role in beta(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 beta(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-DeltaLIELD) or beta(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 beta(2)-adrenergic receptor, while arrestin/beta(2)-adaptin interaction appeared less critical. Arrestin-2 mutants defective in both clathrin and beta(2)-adaptin binding functioned as effective dominant negatives in HEK293 cells and significantly attenuated beta(2)-adrenergic receptor internalization. These mutants should prove useful in better defining the role of arrestins in mediating receptor trafficking.
authors.affiliation: Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA.
authors.forename: You-Me
authors.initials: YM
authors.lastname: Kim
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Benovic
mesh_terms: D033966:Adaptor Protein Complex beta Subunits; D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000737:chemistry / Q000502:physiology*; D001665:Binding Sites; D001692:Biological Transport; D019556:COS Cells; D002417:Cattle; D002966:Clathrin / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000502:physiology*; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*
chemical_list: D033966:Adaptor Protein Complex beta Subunits; D019390:Arrestins; D002966:Clathrin; D008565:Membrane Proteins; D010750:Phosphoproteins; D018343:Receptors, Adrenergic, beta-2