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Modulation of the arrestin-clathrin interaction in cells. Characterization of beta-arrestin dominant-negative mutants
J G Krupnick1, F Santini, A W Gagnon
1Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Abstract:
We recently demonstrated that nonvisual arrestins interact via a C-terminal binding domain with clathrin and function as adaptor proteins to promote beta2-adrenergic receptor (beta2AR) internalization. Here, we investigated the potential utility of a mini-gene expressing the clathrin-binding domain of beta-arrestin (beta-arrestin (319-418)) to function as a dominant-negative with respect to beta2AR internalization and compared its properties with those of beta-arrestin and beta-arrestin-V53D, a previously reported dominant-negative mutant. In vitro studies demonstrated that beta-arrestin-V53D bound better to clathrin than beta-arrestin but was significantly impaired in its interaction with phosphorylated G protein-coupled receptors. In contrast, whereas beta-arrestin (319-418) also bound well to clathrin it completely lacked receptor binding activity. When coexpressed with the beta2AR in HEK293 cells, beta-arrestin (319-418) effectively inhibited agonist-promoted receptor internalization, whereas beta-arrestin-V53D was only modestly effective. However, both constructs significantly inhibited the stimulation of beta2AR internalization by beta-arrestin in COS-1 cells. Interestingly, immunofluorescence microscopy analysis reveals that both beta-arrestin (319-418) and beta-arrestin-V53D are constitutively localized in clathrin-coated pits in COS-1 cells. These results indicate the potential usefulness of beta-arrestin (319-418) to effectively block arrestin-clathrin interaction in cells and suggest that this construct may prove useful in further defining the mechanisms involved in G protein-coupled receptor trafficking.
Insights
A novel mini-gene, beta-arrestin (319-418), effectively blocks beta2-adrenergic receptor internalization by inhibiting arrestin-clathrin interactions. This tool aids in understanding G protein-coupled receptor trafficking mechanisms.
Area of Science:
- Cell biology
- Molecular pharmacology
- Biochemistry
Background:
- Nonvisual arrestins act as adaptor proteins, interacting with clathrin via their C-terminal domain to facilitate beta2-adrenergic receptor (beta2AR) internalization.
- Previous studies identified beta-arrestin-V53D as a dominant-negative mutant affecting beta2AR internalization.
Purpose of the Study:
- To investigate the utility of a mini-gene expressing the clathrin-binding domain of beta-arrestin (beta-arrestin (319-418)) as a dominant-negative inhibitor of beta2AR internalization.
- To compare the properties of beta-arrestin (319-418) with full-length beta-arrestin and the mutant beta-arrestin-V53D.
Main Methods:
- In vitro binding assays to assess interactions between arrestin constructs, clathrin, and phosphorylated receptors.
- Coexpression of beta-arrestin constructs with beta2AR in HEK293 and COS-1 cells to evaluate inhibition of receptor internalization.
- Immunofluorescence microscopy to determine the cellular localization of arrestin constructs.
Main Results:
- Beta-arrestin (319-418) bound clathrin effectively but lacked receptor binding activity.
- Beta-arrestin (319-418) significantly inhibited agonist-promoted beta2AR internalization in HEK293 cells, outperforming beta-arrestin-V53D.
- Both beta-arrestin (319-418) and beta-arrestin-V53D were constitutively localized in clathrin-coated pits and inhibited beta-arrestin-stimulated beta2AR internalization in COS-1 cells.
Conclusions:
- The beta-arrestin (319-418) mini-gene serves as an effective tool to block arrestin-clathrin interactions, thereby inhibiting beta2AR internalization.
- This construct holds potential for further elucidating the mechanisms governing G protein-coupled receptor trafficking.