Modulation of the arrestin-clathrin interaction in cells. Characterization of beta-arrestin dominant-negative mutants.
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.
Krupnick
J G
JG
Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Santini
F
F
Gagnon
A W
AW
Keen
J H
JH
Benovic
J L
JL
D000818:Animals; D019393:Arrestin / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D002417:Cattle; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis; D019204:GTP-Binding Proteins / Q000378:metabolism; D005799:Genes, Dominant; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism
D019393:Arrestin; D002966:Clathrin; D018343:Receptors, Adrenergic, beta-2; D019204:GTP-Binding Proteins