Upon cleavage by calpain, the “soluble” AC domain migrates to different subcellular organelles.

<p>Subcellular localization of the “soluble” AC domain as determined by confocal microscopy. Incubation of J774A.1 cells with ACT (35 nM) results in a colocalization of the “soluble” AC domain with the nucleus and the mitochodria after 10 min. Migration is prevented by pretreatment of cells with calpeptin (100 µM). Cells were preincubated for 30 min with the inhibitor prior to ACT addition, then incubated for 10 min with the toxin, washed three times, fixed and permeabilized to analyze AC domain by immunohistochemistry (A). Z-stack images obtained in the same conditions as in panel A, showing colocalization of “soluble” AC domain with the nucleus and mitochondria, and prevention of migration by pretreatment of cells with calpeptin (100 µM). Anti-nucleoporin p62 staining shows no colocalization of “soluble” AC domain with the nuclear envelope (B). Detection of the “soluble” AC domain into isolated nuclei, mitochondria or ER by Western blot analysis. Nuclear, ER and mitochondrial fractions isolated from cells treated with ACT (35 nM) for 10 min were analyzed by Western blot and stained with the anti-AC MAb 3D1. Anti-histone H2B antibody and anti-VDAC/porin antibody were used to stain histone H2B and the VDAC/porin as molecular markers of the nuclei and mitochondria, respectively. Calregulin was used as marker for ER fractions (C). Adenylate cyclase activity is enhanced very significantly both in nuclear extracts and mitochondrial fractions obtained from 35 nM ACT treated cells. cAMP production was determined as described in <i>Materials and Methods</i> (D).