Calnuc, an EF-Hand Ca~2+ binding protein, specifically interacts with the C-terminal αalfa5-helix of Gαi3

摘要

Calnuc (nucleobindin) was previously shown to be present both in the cytosol and in the Golgi and to be the major Golgi Ca~2+ 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 pulses chase experiments. the two pools of calnuc had different mobili- ties, 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-immu- noprecipitated from NlH 3T3 cells stably overexpressing either activated (Q204L) or inactivated (G203A) Gαi3. Binding was Ca~2+ and Mg~2+-dependent. CaInuc 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 coiIed-coiI domain constituted by the two EF-hands. binds to Gαi3's C-terminal cα5-helix. These results demonstrate that calnuc may play an important role in G protein- and Ca'+-regulated signal transduction events.