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. 2008 Jun 6;283(23):15903–15911. doi: 10.1074/jbc.M710006200

FIGURE 7.

FIGURE 7.

Specificity determinants within β-arrestin-2 for binding to JNK3. A, schematic of the β-arrestin constructs used in the experiments and a summary of their binding to JNK3 (- represents weak or no binding; + represents strong binding). The position of the D-domain like sequence is indicated. B and C, constructs expressing GST-tagged β-arrestin-2 deletion mutants were introduced into COS-7 cells together with expression vectors for HA-JNK3 (B) and HA-ASK1 (C). GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads, and the JNK3 or ASK1 present in the pull-down (PD) was examined by immunoblot using anti-HA antibody. The expression of the ASK1, JNK3, and β-arrestin-2 was examined by immunoblotting the lysates with anti-HA and anti-GST antibodies, respectively. D and E, constructs expressing the indicated GST-tagged β-arrestin proteins were introduced into COS-7 cells together with an expression vector for HA-JNK3. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads and the JNK3 present in the pull-down (PD) was examined by immunoblot using anti-HA antibody. The expression of the JNK3 and the β-arrestin proteins was examined by immunoblotting the lysates with anti-HA and anti-GST antibodies, respectively. F, sequence alignment of the putative D-domains in β-arrestin-1 and β-arrestin-2. The D-domains are boxed, and the key Ser and Pro residues highlighted. Numbers refer to the amino acid position. G, constructs expressing the indicated FLAG-tagged β-arrestin-2 mutants were introduced into COS-7 cells together with an expression vector for either GST or GST-JNK3. GST-containing complexes were isolated from cell lysates with glutathione-Sepharose beads, and the β-arrestin proteins present in the pull-down (PD) was examined by immunoblot using the M2 antibody. The expression of the JNK3 and the β-arrestin proteins was examined by immunoblotting the lysates with anti-GST and M2 antibodies, respectively. H, constructs expressing the indicated FLAG-tagged β-arrestin proteins were introduced into COS-7 cells together with an expression vector for GST-JNK3. FLAG-β-arrestin-2 complexes were isolated from cell lysates by immunoprecipitation with the M2 antibody (M2-IP), and JNK3 present in the precipitates was examined by immunoblot using an anti-GST antibody. The expression of the JNK3 and the β-arrestin proteins was examined by immunoblotting the lysates with anti-GST and M2 antibodies, respectively. I, scheme of β-arrestin-2 scaffold assembly. The C terminus of β-arrestin-2 (denoted C) binds to the extended N terminus of JNK3 (denoted N), and this binding is controlled by residues within the D-domain of β-arrestin-2 (denoted D). MKK4 is recruited to the complex via its D-domain (denoted D) binding to JNK3. ASK1 binds to the N terminus of β-arrestin-2 (denoted N). Experiments were performed either two or three times, and representative immunoblots are shown.