beta-Arrestin/AP-2 interaction in G protein-coupled receptor internalization: identification of a beta-arrestin binging site in beta 2-adaptin.
beta-Arrestins, proteins involved in the turn-off of G protein-coupled receptor (GPCR) activation, bind to the beta(2)-adaptin subunit of the clathrin adaptor AP-2. The interaction of beta(2)-adaptin with beta-arrestin involves critical arginine residues in the C-terminal domain of beta-arrestin and plays an important role in initiating clathrin-mediated endocytosis of the beta(2)-adrenergic receptor (beta(2)AR) (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 beta-arrestin-binding site in beta(2)-adaptin has not been identified, and little is known about the role of beta-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 beta(2)-adaptin that are important in beta-arrestin binding. These residues are located in the platform subdomain of the C terminus of beta(2)-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 beta-arrestin/AP-2/clathrin complex in the endocytosis of GPCRs such as the beta(2)AR and vasopressin type II receptor was evaluated using mutant constructs of the beta(2)-adaptin C terminus containing either the clathrin and the beta-arrestin binding domains or the beta-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 beta(2)AR and the vasopressin type II receptor. In addition, although the beta(2)-adaptin construct containing both the clathrin and beta-arrestin binding domains was able to block the endocytosis of transferrin receptors, a beta(2)-adaptin construct capable of associating with beta-arrestin but lacking its high affinity clathrin interaction did not interfere with transferrin receptor endocytosis. These results suggest that the interaction of beta-arrestin with beta(2)-adaptin represents a selective endocytic trigger for several members of the GPCR family.
Laporte
Stephane A
SA
Howard Hughes Medical Institute Laboratories, the Department of Cell Biology, and the Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Miller
William E
WE
Kim
Kyeong-Man
KM
Caron
Marc G
MG
D033966:Adaptor Protein Complex beta Subunits; D019390:Arrestins / Q000378:metabolism*; D001665:Binding Sites; D002478:Cells, Cultured; D022163:Clathrin-Coated Vesicles / Q000502:physiology; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004705:Endocytosis; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D000071557:beta-Arrestins
D033966:Adaptor Protein Complex beta Subunits; D019390:Arrestins; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D000071557:beta-Arrestins; D019204:GTP-Binding Proteins