title: Ca(2+) -calmodulin interacts with DdCAD-1 and promotes DdCAD-1 transport by contractile vacuoles in Dictyostelium cells.
abstract: The Ca(2+) -dependent cell-cell adhesion molecule DdCAD-1, encoded by the cadA gene of Dictyostelium discoideum, is synthesized at the onset of development as a soluble protein and then transported to the plasma membrane by contractile vacuoles. Calmodulin associates with contractile vacuoles in a Ca(2+) -dependent manner, and co-localizes with DdCAD-1 on the surface of contractile vacuoles. Bioinformatics analysis revealed multiple calmodulin-binding motifs in DdCAD-1. Co-immunoprecipitation and pull-down studies showed that only Ca(2+) -bound calmodulin was able to bind DdCAD-1. Structural integrity of DdCAD-1, but not the native conformation, was required for its interaction with calmodulin. To investigate the role of calmodulin in the import of DdCAD-1 into contractile vacuoles, an in vitro import assay consisting of contractile vacuoles derived from cadA(-) cells and recombinant proteins was employed. Prior stripping of the bound calmodulin from contractile vacuoles by EGTA impaired import of DdCAD-1, which was restored by addition of exogenous calmodulin. The calmodulin antagonists W-7 and compound 48/80 blocked the binding of calmodulin onto stripped contractile vacuoles, and inhibited the import of DdCAD-1. Together, the data show that calmodulin forms a complex with DdCAD-1 and promotes the docking and import of DdCAD-1 into contractile vacuoles.
  CaM physically interacts with DdCAD-1 by pull down (View Interaction: 1, 2) DdCAD-1 binds to CaM by far western blotting (View interaction) DdCAD-1 physically interacts with CaM by anti bait coimmunoprecipitation (View interaction).
authors: Department of Biochemistry, University of Toronto, Ontario, Canada.
authors: Shrivani
authors: S
authors: Sriskanthadevan
authors: Simuran K
authors: SK
authors: Brar
authors: Kumararaaj
authors: K
authors: Manoharan
authors: Chi-Hung
authors: CH
authors: Siu
mesh_terms: D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000502:physiology*; D002147:Calmodulin / Q000502:physiology*; D015815:Cell Adhesion Molecules / Q000502:physiology*; D004023:Dictyostelium / Q000378:metabolism*; D047468:Immunoprecipitation; D014617:Vacuoles / Q000378:metabolism*
chemical_list: D002135:Calcium-Binding Proteins; D002147:Calmodulin; D015815:Cell Adhesion Molecules; C100604:DdCAD-1 protein, Dictyostelium discoideum; D002118:Calcium