Fig. 5.

Role of four calmodulin binding domains (PB1-3, CB4) in gravin redistribution and subcellular localization. (A, B) Comparison of the rate of redistribution from cell membrane to cytosol for either WT gravin-EGFP and (ΔPB1-3) gravin-EGFP (A) or WT gravin-EGFP and (ΔPB1-3+) gravin-EGFP (B) after ionomycin treatment. The deleted regions are illustrated below the graphs. No difference was observed between WT gravin and (ΔPB1-3) gravin, but the rate of (ΔPB1-3+) gravin translocation from membrane to cytosol was significantly reduced compared to WT gravin. (Comparison at each time point revealed significant differences between constructs as indicated by the horizontal bar; Mann-Whitney Rank Sum Test, p < 0.05). (C) Graph illustrating the effect of deleting the CB4 region on localization of gravin at the cell periphery in transfected cells. Note that localization of the (ΔPB1-3, CB4) mutant at the cell cortex was significantly reduced compared to WT gravin. Reconstitution of the CB4 domain (ΔPB1-3+) restored membrane localization, while deletion of the CB4 domain (ΔCB4) alone resulted in a decrease in membrane localization similar to that of ΔPB1-3,CB4. Astericks indicate significant differences from WT gravin-EGFP (one-way ANOVA followed by a Holm-Sidak post-hoc test; p < 0.05). (D) A Western blot demonstrating the expression of full-length gravin-EGFP and its deletion mutants in AN3CA cells. Sixty micrograms total protein was loaded in all lanes. The number of amino acids comprising each construct is marked at the top of the blot.