Identification of essential residues in the AT1R CT for tubulin interaction.
(A) Sequence of the AT1R CT containing four Lys residues at positions 307, 308, 310 and 311. (B) Effect of mutating the four Lys residues to Glu on the AT1R CT interaction with tubulin. GST and GST fusion proteins encoding the AT1R CT or its mutant in which the four Lys residues were mutated to Glu (4K-4E) were incubated with rat brain cytosolic extracts (top panel) or purified tubulin (middle panel) as described in the legend of Fig. 1B. Bottom panels: Coomassie Brilliant Blue (CBB) staining of purified GST fusion proteins after SDS-PAGE. (C) Quantitative data of (B). (D) Effect of mutating individual Lys residue on the AT1R CT interaction with purified tubulin. GST and GST fusion proteins encoding the AT1R CT or its mutants in which Lys residues at positions 307, 308, 310 and 311 were individually mutated to Glu were incubated with purified tubulin as described in the legend of Fig. 1B. E, Quantitative data of (D). In (C) and (E), quantitative data are expressed as percentages of tubulin interacting with the AT1R CT and presented as the mean ± S.E. of at least three individual experiments. *, p < 0.05 versus the AT1R CT.