The overexpression of WT-RhoGDI1 or S34D-RhoGDI1 inhibits CCK-induced amylase secretion whereas S96D-RhoGDI1 does not.

<p><b>A)</b> Isolated pancreatic acini expressing β-Gal, WT-RhoGDI1 and the phosphomimetic mutants S34D and S96D were stimulated with different concentrations of CCK for 30 min, and amylase release was measured. <b>RhoGDI1 plays a negative regulatory role on amylase secretion by sequestering inactive RhoA</b>. <b>B)</b> CA-RhoA, CA-Rac1 and WT-RhoGDI1 were co-expressed in pancreatic acini. Isolated pancreatic acini expressing WT-RhoGDI1, CA-RhoA, CA-Rac1 or the combination of both were stimulated with different concentration of CCK for 30 min, and amylase release was measured. The expression of CA-RhoA (left panel) reversed the inhibitory effect of overexpressed WT-RhoGDI1 on amylase secretion, whereas the expression of CA-Rac1 (right panel) does not. Values are means ± SE (n = 3–4 experiments) of amylase release expressed as a percentage of total. * p<0.05 vs. control acini; † p<0.05 vs. RhoGDI1. <b>Active RhoA and active Rac1 do not interact with RhoGDI1</b>. <b>C)</b> The association between CA-RhoA or CA-Rac1 with RhoGDI1 was analyzed in total lysates using co-immunoprecipitation. The expression of CA-RhoA, CA-Rac1 and WT-RhoGDI1 was analyzed by Western-blotting using anti-HA-tag and anti-RhoGDI1 antibodies, respectively. A representative immunoblot shows that RhoGDI1 does not form a complex with CA-RhoA or CA-Rac1. Comparable amount of immunoprecitated RhoGDI1 was confirmed by Western-blotting using anti-RhoGDI1 antibody. Data are representative of 4 independent experiments; the dissociation levels were normalized to control = 1.0 in each assay and expressed as fold change.