Figure 2.

Interaction between βarrestin 2 and individual subunits of AP-2 or clathrin as assessed by β-galactosidase activity in yeast. Results of colony-lift filter assays, colony growth assays on adenine-deficient medium, and liquid culture β-galactosidase assays are shown. (a) The binding interactions between βarrestin 2 (βarr2) and each of the four subunits of AP-2 were analyzed in yeast cells transformed with plasmids encoding GAL4 BD-βarr2 and one of the various GAL4 AD-adaptin subunits (α, β2, μ2, and σ2). (b) Results of the expression of the lacZ reporter gene and adenine growth in yeast cells transformed with GAL4 BD-βarr2, -βarr2_AAEA, or different fragments of βarr2 with the GAL4 AD-β2-adaptin subunits. Only transformants expressing interacting proteins were able to grow in the absence of adenine, eliminating the contribution of transformants with intrinsic activity (ia). Positive results for β-galactosidase activity or growth on adenine-deficient plates are indicated by (+), and no interaction is indicated by (−). (c) The interaction of βarr2 or βarrestin2 clathrin-binding deficient mutant (βarr2_AAEA) with GAL4 AD-clathrin (Clath) or GAL4 AD-β2-adaptin (β) was examined. A more intense blue coloration on the filter indicates a stronger binding interaction between the two proteins. Results, expressed in relative light units (RLU), are the mean ± SD of triplicate determinations. All results are representative of four to six independent experiments.