Abstract
Calnuc (nucleobindin) was previously shown to be present both in the cytosol and in the Golgi and to be the major Golgi Ca2+ binding protein. In this study we verified the existence of the cytosolic pool of calnuc and investigated its interaction with Gαi3. Cytosolic calnuc was released by mild digitonin permeabilization. In pulse–chase experiments, the two pools of calnuc had different mobilities, suggesting different posttranslational modifications. That calnuc interacts with Gαi3 in vivo was verified by the finding that Gαi3 could be crosslinked intracellularly to calnuc and co-immunoprecipitated from NIH 3T3 cells stably overexpressing either activated (Q204L) or inactivated (G203A) Gαi3. Binding was Ca2+ and Mg2+-dependent. Calnuc and Gαi3-GFP codistributed primarily in the Golgi region. By yeast two-hybrid analysis, the binding site on Gαi3 for calnuc was mapped to the C-terminal region because removal of the last 12 amino acids (but not 11) abolished the interaction. Peptide competition indicated that calnuc, with its coiled-coil domain constituted by the two EF-hands, binds to Gαi3's C-terminal α5-helix. These results demonstrate that calnuc may play an important role in G protein- and Ca2+-regulated signal transduction events.