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First published online 11 July 2006
doi: 10.1242/jcs.03052

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An essential role for endocytosis of rhodopsin through interaction of visual arrestin with the AP-2 adaptor

Department of Biological Sciences, Dartmouth College, Hanover, NH 03755, USA

View larger version (132K): [in a new window]   Fig. 1. The AP-2 adaptor complex is necessary for the norpA-induced endocytosis of stable rhodopsin-arrestin complexes. All panels show cross sections (0.75 µm) from a frozen Drosophila retina stained with both arrestin and rhodopsin antibodies followed by fluorescein- and rhodamine-labeled secondary antibodies respectively. (A-C) Wild-type, (D-F) norpA and (G-I) norpA;-adaptin. The flies were dark-reared and then treated with constant room light for 24 hours prior to fixation. Dissected eyes were fixed and sectioned as described in Materials and Methods. A, D and G were stained with arrestin-specific antibodies, B, E and H were stained with rhodopsin-specific antibodies, C, F and I are merged images.

View larger version (128K): [in a new window]   Fig. 2. The AP-2 adaptor complex is necessary for norpA-mediated retinal degeneration. All panels show cross sections (0.5 µm) of retinas from (A) wild-type, (B) norpA, (C) -adaptin and (D) norpA;-adaptin flies. The flies were dark-reared and then exposed to 5 days of constant room light prior to fixation. Tissue was fixed and embedded as described in Materials and Methods.

View larger version (34K): [in a new window]   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.

View larger version (115K): [in a new window]   Fig. 4. Point mutations in the AP-2 binding domain of arrestin do not cause rapid light-dependent degeneration. All panels are cross sections (0.5 µm) of Drosophila retinas from (A) wild-type, (B) arr2null, (C) arr2K391A and (D) arr2R393A flies. Dark reared flies were treated with 5 days of constant room light then fixed and sectioned as described in Materials and Methods.

View larger version (137K): [in a new window]   Fig. 5. An intact AP-2 binding domain in arrestin is necessary for norpA-induced endocytosis of persistent rhodopsin-arrestin complexes. Cross sections (0.75 µm) from a frozen Drosophila retina were stained with both arrestin and rhodopsin antibodies followed by fluorescein and rhodamine-labeled secondary antibodies, respectively. (A-C) norpA, (D-F) norpA; arr2K391A and (G-I) norpA; arr2R393A. The flies were dark-reared and then treated with constant room light for 24 hours prior to fixation. Dissected eyes were fixed and sectioned as described in Materials and Methods. A, D and G were stained with arrestin-specific antibodies, B, E and H were stained with rhodopsin-specific antibodies, C, F, and I are merged images.

View larger version (132K): [in a new window]   Fig. 6. The interaction between arrestin and AP-2 is required for norpA-mediated retinal degeneration. All panels show cross sections (0.5 µm) of Drosophila retinas from (A) wild-type, (B) norpA, (C) norpA; arr2K391A and (D) norpA; arr2R393A. Dark-reared flies were treated with 5 days of constant room light then fixed and sectioned as described in Materials and Methods.

View larger version (114K): [in a new window]   Fig. 7. Mutations in the AP-2 binding domain of arrestin cause slow retinal degeneration. All panels show cross sections (0.5 µm) of Drosophila retinas from (A) wild-type, (B) arr25, (C) arr2K391A and (D) arr2R393A flies. Dark-reared flies were treated for 30 days of 12 hours light/12 hours dark, then fixed and embedded as described in Materials and Methods.