Krupnick J G, Santini F, Gagnon A W, Keen J H, Benovic J L
Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Biol Chem. 1997 Dec 19;272(51):32507-12. doi: 10.1074/jbc.272.51.32507.
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.
我们最近证明，非视觉抑制蛋白通过C末端结合结构域与网格蛋白相互作用，并作为衔接蛋白促进β2肾上腺素能受体（β2AR）的内化。在此，我们研究了一种表达β抑制蛋白网格蛋白结合结构域的微型基因（β抑制蛋白（319 - 418））在β2AR内化方面作为显性负性分子的潜在效用，并将其特性与β抑制蛋白和β抑制蛋白-V53D（一种先前报道的显性负性突变体）的特性进行了比较。体外研究表明，β抑制蛋白-V53D与网格蛋白的结合比β抑制蛋白更好，但与磷酸化G蛋白偶联受体的相互作用明显受损。相比之下，虽然β抑制蛋白（319 - 418）也能很好地与网格蛋白结合，但其完全缺乏受体结合活性。当与β2AR在HEK293细胞中共表达时，β抑制蛋白（319 - 418）有效抑制激动剂促进的受体内化，而β抑制蛋白-V53D的效果则不太显著。然而，这两种构建体在COS-1细胞中均显著抑制β抑制蛋白对β2AR内化的刺激作用。有趣的是，免疫荧光显微镜分析显示，β抑制蛋白（319 - 418）和β抑制蛋白-V53D在COS-1细胞中均组成性地定位于网格蛋白包被小窝中。这些结果表明β抑制蛋白（319 - 418）在有效阻断细胞中抑制蛋白-网格蛋白相互作用方面具有潜在用途，并表明该构建体可能有助于进一步阐明G蛋白偶联受体转运所涉及的机制。
