Figure 1.
The PDZ domain of erbin binds to the C-terminal domain of α11.3. A, Schematic of rat α11.3 showing class I PDZ-binding consensus sequence (ITTL) in the cytoplasmic C-terminal domain and the region used for GST-α11.3 CT in pull-down assays (amino acids 1962–2155). B, Schematic of mouse erbin indicating position of LRR (amino acids 392–428) and the PDZ domain (amino acids 1280–1371). C, Binding of erbin to α11.3 CT. GST-α11.3 CT (lane 3) or GST alone (lane 2) was immobilized on glutathione-agarose beads and incubated with lysates from cells transfected with GFP-erbin965 (amino acids 965–1371). Bound GFP-erbin965 was detected by Western blot with anti-GFP antibodies. Lane 1 shows ∼15% of GFP-erbin965 input used in the pull-down assay. D, Binding of erbinPDZ but not MAGI1PDZ1 to α11.3 CT. GST-α11.3 CT was incubated with purified his-S-erbinPDZ (lanes 3–5) or his-S-MAGI1PDZ1 (lanes 6–8). Input his-S-tagged protein was the following (in μg): 1 (lanes 3, 6), 2 (lanes 4, 7), and 4 (lanes 5, 8). Input lanes 1 and 2 show his-S-tagged proteins used in the assay (1 μg). E, Impaired binding of erbinPDZ to α11.3 CTL–A. His-S-erbinPDZ protein (lanes 1, 4, 1.5 μg; lanes 2, 5, 3 μg; lanes 3, 6, 6 μg) was incubated with GST-α11.3 CT (lanes 1–3) or GST-α11.3 CTL–A (lanes 4–6). In D and E, bound proteins were detected by Western blot (top) with anti-S-protein antibodies, and Ponceau staining (bottom) shows equal levels of GST-α11.3 CT or GST-α11.3 CTL–A in each group.