Calnuc, an EF-hand Ca(2+) binding protein, specifically interacts with the C-terminal alpha5-helix of G(alpha)i3.
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 G(alpha)i3. 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 G(alpha)i3 in vivo was verified by the finding that G(alpha)i3 could be crosslinked intracellularly to calnuc and co-immunoprecipitated from NIH 3T3 cells stably overexpressing either activated (Q204L) or inactivated (G203A) G(alpha)i3. Binding was Ca(2+) and Mg(2+)-dependent. Calnuc and G(alpha)i3-GFP codistributed primarily in the Golgi region. By yeast two-hybrid analysis, the binding site on G(alpha)i3 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 G(alpha)i3's C-terminal alpha5-helix. These results demonstrate that calnuc may play an important role in G protein- and Ca(2+)-regulated signal transduction events.
Lin
P
P
Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093-0651, USA.
Fischer
T
T
Weiss
T
T
Farquhar
M G
MG
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006133:Growth Substances / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008274:Magnesium / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D011485:Protein Binding / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017384:Sequence Deletion
D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; D006133:Growth Substances; D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; D000080866:Nucleobindins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D008274:Magnesium; D002118:Calcium