The interaction of beta-arrestin with the AP-2 adaptor is required for the clustering of beta 2-adrenergic receptor into clathrin-coated pits.
Beta-arrestins are cytosolic proteins that regulate the signaling and the internalization of G protein-coupled receptors (GPCRs). Although termination of receptor coupling requires beta-arrestin binding to agonist-activated receptors, GPCR endocytosis involves the coordinate interactions between receptor-beta-arrestin complexes and other endocytic proteins such as adaptor protein 2 (AP-2) and clathrin. Clathrin interacts with a conserved motif in the beta-arrestin C-terminal tail; however, the specific molecular determinants in beta-arrestin that bind AP-2 have not been identified. Moreover, the respective contributions of the interactions of beta-arrestin with AP-2 and clathrin toward the targeting of GPCRs to clathrin-coated vesicles have not been established. Here, we identify specific arginine residues (Arg(394) and Arg(396)) in the beta-arrestin 2 C terminus that mediate beta-arrestin binding to AP-2 and show, in vitro, that these domains in beta-arrestin 1 and 2 interact equally well with AP-2 independently of clathrin binding. We demonstrate in HEK 293 cells by fluorescence microscopy that beta(2)-adrenergic receptor-beta-arrestin complexes lacking the beta-arrestin-clathrin binding motif are still targeted to clathrin-coated pits. In marked contrast, receptor-beta-arrestin complexes lacking the beta-arrestin/AP-2 interactions are not effectively compartmentalized in punctated areas of the plasma membrane. These results reveal that the binding of a receptor-beta-arrestin complex to AP-2, not to clathrin, is necessary for the initial targeting of beta(2)-adrenergic receptor to clathrin-coated pits.
Laporte
S A
SA
Howard Hughes Medical Institute Laboratories and the Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Oakley
R H
RH
Holt
J A
JA
Barak
L S
LS
Caron
M G
MG
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D019390:Arrestins / Q000737:chemistry / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D000071557:beta-Arrestins
D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D019390:Arrestins; D002966:Clathrin; D008565:Membrane Proteins; D018343:Receptors, Adrenergic, beta-2; D000071557:beta-Arrestins