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Fig. 3. Point mutations in the AP-2 binding domain of Arr2 do not inhibit the binding of arrestin to rhodopsin or the ability of the mutant arrestins to regulate the phototransduction cascade. (A) An alignment of ß and visual arrestins. The `clathrin box' found in ßarr1 and 2 are in bold and italicized. The AP-2 binding domain is in bold and underlined. The two residues that were mutated for this study are marked with an asterisk. (B) Head lysates from wild-type, arr2K391A and arr2R393A flies were generated and incubated with a maltose binding protein-AP-2ß Ear domain fusion protein. Lanes 1-3 show represent head equivalents from the lysate. Lanes 4-6 represent Arr2 protein bound to the AP-2ß Ear domain. wt, wild-type; K, arr2K391A; R, arr2R393A. Overexposure of the blot labeled `bound' failed to show any significant binding of the K or R point mutants (data not shown) (C) Western blot of fractionated fly head lysates. Dark-reared flies were treated with either 2 seconds of blue light or 2 seconds of blue light followed by 4 seconds of orange light. wt, wild-type; K, arr2K391A; R, arr2R393A. Pellet (P) and supernatant (S) fractions were subjected to SDS-PAGE and western blot analysis. (D) Quantification of the percentage of arrestin bound to rhodopsin-containing membranes. The data shown are the averages of five independent experiments. For the blue light treatment the following percentages of arrestin were bound, wild type 89±9%, arr2K391A 95±5% and arr2R393A 99%±1%. For the blue/orange light treatment the following percentages of arrestin were bound, wild-type 27±10%, arr2K391A 25±19% and arr2R393A 52±20%. Data are means ± s.d. (E) Electroretinogram analysis of wild-type, arr25, arr2K391A and arr2R393A dark reared newly eclosed flies. The flies were treated with 1 second of 480 nm (blue) light and the total electrical response of the eye was measured.
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