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. 2006 Jul 28;281(30):21491-21499.
doi: 10.1074/jbc.M603659200. Epub 2006 May 31.

Visual and both non-visual arrestins in their "inactive" conformation bind JNK3 and Mdm2 and relocalize them from the nucleus to the cytoplasm

Xiufeng Song  1 , Dayanidhi Raman  1 , Eugenia V Gurevich  1 , Sergey A Vishnivetskiy  1 , Vsevolod V Gurevich  2
Affiliations

Visual and both non-visual arrestins in their "inactive" conformation bind JNK3 and Mdm2 and relocalize them from the nucleus to the cytoplasm

Xiufeng Song et al. J Biol Chem. .

Abstract

Arrestins bind active phosphorylated G protein-coupled receptors, terminating G protein activation. Receptor-bound non-visual arrestins interact with numerous partners, redirecting signaling to alternative pathways. Arrestins also have nuclear localization and nuclear exclusion signals and shuttle between the nucleus and the cytoplasm. Constitutively shuttling proteins often redistribute their interaction partners between the two compartments. Here we took advantage of the nucleoplasmic shuttling of free arrestins and used a "nuclear exclusion assay" to study their interactions with two proteins involved in "life-and-death" decisions in the cell, the kinase JNK3 and the ubiquitin ligase Mdm2. In human embryonic kidney 293 cells green fluorescent protein (GFP)-JNK3 and GFP-Mdm2 predominantly localize in the nucleus, whereas visual arrestin, arrestin2(Q394L) mutant equipped with the nuclear exclusion signal, and arrestin3 localize exclusively to the cytoplasm. Coexpression of arrestins moves both GFP-JNK3 and GFP-Mdm2 to the cytoplasm. Arrestin mutants "frozen" in the basal conformation are the most efficacious. Thus, arrestins in their basal state interact with JNK3 and Mdm2, suggesting that arrestins are likely "preloaded" with their interaction partners when they bind the receptor. Robust interaction of free arrestins with JNK3 and Mdm2 and their ability to regulate subcellular localization of these proteins may play an important role in the survival of photoreceptors and other neurons, as well as in retinal and neuronal degeneration.

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Figures

FIGURE 1
FIGURE 1. Differential subcellular distribution of endogenous arrestins in different types of neurons
Sections of the brain (A–D) or retina (E–G) were labeled immunohistochemically with arrestin subtype-specific antibodies and counterstained with 4′,6-diamidino-2-phenylindole to visualize nuclei as described under “Materials and Methods.” A, C, and F, overlays of immunostaining for arrestins (green) and 4′,6-diamidino-2-phenylindole staining of nuclei (blue).B,D, E and G, the corresponding images of arrestin staining. A and B, high power photomicrographs of the arrestin2 subcellular distribution in the layer V pyramidal cells of the rat primary somatosensory cortex. Note the high concentration of arrestin2 in the nuclei. C and D, photomicrographs of rat striatal neurons showing mostly cytosolic localization of arrestin2. E–G, subcellular localization of visual arrestin in dark-adapted rod photoreceptors. Low power photomicrograph of the dark-adapted (overnight) retina is shown in panel E. In the dark, visual arrestin is localized to the inner segments (IS) and cell bodies of photoreceptors (outer nuclear layer, ONL), whereas outer segments (OS) of photoreceptors are devoid of arrestin. F and G, high power microphotographs of photoreceptor cells in the outer nuclear layer of the retina demonstrating that visual arrestin is completely excluded from the nuclei.
FIGURE 2
FIGURE 2. Subcellular distribution of wild-type and differentially tagged arrestins in HEK293 cells
The indicated untagged, FLAG-tagged (F), and GFP-tagged arrestins were visualized with F4C1 pan-arrestin antibody (20), M2 anti-FLAG antibody, and native GFP fluorescence, respectively. Note the exclusive (visual and arrestin3) or preferential (arrestin2) cytoplasmic localization of untagged and FLAG-tagged arrestins. The addition of an ~20-kDa GFP tag, as well as incubation with an inhibitor of NES-dependent nuclear export, leptomycin B (LMB) (50 ng/ml), increase the proportion of visual and arrestin2 in the nucleus. Wild-type and FLAG-tagged arrestins 2, 3, and visual were expressed at comparable levels of 74 + 24, 53 + 11, and 196 + 37 pmol/mg of protein, respectively, as measured by quantitative Western blot (100 ng of total protein/lane) with the indicated amount of the corresponding purified proteins as standards. The expression levels of non-visual arrestins are higher than the levels observed in mature neurons (3, 4), whereas for visual arrestin it is lower than its expression in rod photoreceptors (44, 45). Visual arrestin-GFP was expressed at 88 + 21 pmol/mg of protein, whereas GFP fusions of both non-visual arrestins were expressed at 11 + 1 pmol/mg. Here and in the rest of the figures the images of representative cells (of 30–50 cells inspected and scored) are shown. Vis, visual (rod) arrestin; Arr2, arrestin2; Arr3, arrestin3.
FIGURE 3
FIGURE 3. The role of the C-terminal nuclear export signal in the subcellular distribution of non-visual arrestins
The indicated untagged arrestins were visualized in HEK293 cells with F4C1 pan-arrestin antibody (20). The presence (in arrestin3 and the arrestin2(Q394L) mutant) or absence (in arrestin2 and the arrestin3(L394Q) mutant) of the C-terminal NES does not dramatically change the subcellular distribution of untagged arrestins. Arrestin expression levels were the same as in Fig. 2.
FIGURE 4
FIGURE 4. Arrestin-dependent nuclear exclusion of JNK3
GFP-JNK3 was expressed alone or co-expressed with the indicated wild-type and mutant arrestins and visualized by GFP fluorescence. Wild-type visual arrestin, arrestin3, and NES+ arrestin2 relocalize JNK3 from the nucleus to the cytoplasm via an LMB-sensitive mechanism, whereas wild-type arrestin2 does not. Visual arrestin mutants L203C, L280A, and L203C+L280A with one or both putative NES disabled retain their ability to redistribute JNK3 but become LMB insensitive. Disabling the C-terminal NES in arrestin3 by the L394Q mutation only partially inhibits its ability to redistribute JNK3. Arrestin expression levels were the same as in Fig. 2.
FIGURE 5
FIGURE 5. Arrestin interaction with JNK3 does not depend on arrestin conformation
Three forms of each arrestin were co-expressed with GFP-JNK3: wild type (WT), C-terminal triple alanine mutants with a loose active-like conformation (3A), and the mutants frozen in the basal conformation by the deletion of seven residues in the inter-domain hinge region (D7). GFP-JNK3 was visualized by intrinsic fluorescence (green); the indicated FLAG-tagged arrestins were visualized with anti-FLAG antibody (red). Typical cells from two independent experiments (with >20 cells inspected in each) are shown. In every cell that had detectable arrestin expression all three forms of visual arrestin, arrestin2(NES+), and arrestin3 effectively relocalized JNK3 to the cytoplasm. Arrestin expression levels (in pmol/mg of protein) were: visual WT, 240 + 30; 3A, 288 + 28; D7, 55 + 15; arrestin2 WT, 65 + 26; 3A, 162 + 69; D7, 66 + 12; arrestin3 WT, 47 + 9; 3A, 2.7 + 1.8; D7, 12 + 6.
FIGURE 6
FIGURE 6. Mdm2 preferentially interacts with arrestin frozen in the basal conformation
Three forms of visual arrestin, arrestin2(NES+), and arrestin3 were co-expressed with GFP-Mdm2: wild type (WT), C-terminal triple alanine mutant with a loose active-like conformation (3A), and the mutant frozen in the basal conformation by the deletion of seven residues in the inter-domain hinge region (D7). GFP-Mdm2 was visualized by intrinsic fluorescence (green); the indicated FLAG-tagged arrestins were visualized with anti-FLAG antibody (red). The D7 mutant effectively moves Mdm2 to the cytoplasm at relatively low (L) and high (H) expression levels; WT arrestin is less effective, whereas the effect of arrestin-3A mutants can only be observed in cells expressing them at very high levels. Typical cells are shown. Bar graph, for each protein >20 cells expressing arrestin at each (high and low) level, as judged by the intensity of FLAG signal, were scored in each experiment. For each arrestin subtype, the results of three experiments were analyzed by one-way analysis of variance with protein (none, WT, 3A, D7) as a main factor. The effect of protein was significant (p < 0.0001). The significance of the arrestin-induced changes in Mdm2 localization, as revealed by post-hoc analysis (Bonferroni) is indicated: **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (green, as compared with Mdm2 alone; red, as compared with the corresponding WT arrestin). Arrestin expression levels were the same as in Fig. 5.
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