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beta-Arrestin/AP-2 interaction in G protein-coupled receptor internalization: identification of a beta-arrestin
Stephane A Laporte1, William E Miller, Kyeong-Man Kim
1Howard Hughes Medical Institute Laboratories, the Department of Cell Biology, and the Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
Abstract:
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.
Insights
Beta-arrestin binding to beta(2)-adaptin, a component of AP-2, is crucial for G protein-coupled receptor (GPCR) endocytosis. This study identifies specific glutamate residues in beta(2)-adaptin essential for this interaction, revealing a selective mechanism for GPCR internalization.
Area of Science:
- Cell biology
- Molecular biology
- Biochemistry
Background:
- Beta-arrestins regulate G protein-coupled receptor (GPCR) desensitization and internalization.
- Beta-arrestin interaction with the AP-2 complex, specifically beta(2)-adaptin, is critical for clathrin-mediated endocytosis of receptors like the beta(2)-adrenergic receptor (beta(2)AR).
- The precise binding site within beta(2)-adaptin for beta-arrestin and its role in endocytosis of various GPCRs remain largely uncharacterized.
Purpose of the Study:
- To identify the beta-arrestin-binding site on beta(2)-adaptin.
- To investigate the functional significance of the beta-arrestin/AP-2 interaction in the endocytosis of different GPCRs.
- To determine if this interaction is selective for GPCRs.
Main Methods:
- In vitro binding assays to identify key residues in beta(2)-adaptin responsible for beta-arrestin binding.
- Site-directed mutagenesis of beta(2)-adaptin to create constructs with altered binding properties.
- Expression of mutant beta(2)-adaptin constructs in human embryonic kidney 293 cells to assess their impact on receptor internalization.
- Evaluation of the effect of these constructs on the endocytosis of beta(2)AR, vasopressin type II receptor, and transferrin receptor.
Main Results:
- Two glutamate residues (Glu-849 and Glu-902) in the beta(2)-adaptin C-terminal platform subdomain were identified as critical for beta-arrestin binding.
- Mutant beta(2)-adaptin constructs, particularly those binding beta-arrestin, acted as dominant negatives, inhibiting the internalization of beta(2)AR and vasopressin type II receptor.
- A beta(2)-adaptin construct that binds beta-arrestin but not clathrin did not inhibit transferrin receptor endocytosis, suggesting a selective role.
Conclusions:
- The interaction between beta-arrestin and specific glutamate residues in beta(2)-adaptin is essential for the clathrin-mediated endocytosis of certain GPCRs.
- This beta-arrestin/beta(2)-adaptin interaction serves as a selective endocytic trigger for GPCRs, distinct from the general clathrin-mediated endocytosis pathway.
- The findings elucidate a specific mechanism governing GPCR trafficking and offer insights into the regulation of receptor signaling.