Calmodulin associates with the cytoplasmic tail of FcγRIIa. (A) The cytoplasmic tail of human FcγRIIa contains a membrane-proximal, positively charged amino acid sequence analogous to the calmodulin-binding sequence in human GPVI. Identical residues or conservative substitutions are highlighted. (B) Pull-down from human platelet lysates with GST alone or GST-calmodulin (GST-CaM) in the presence of 1mM Ca²⁺. Proteins were captured with glutathione-Sepharose and immunoblotted by using an anti–FcγRIIa tail antiserum. Platelet lysate (PL) was run as a control lane. (C) Washed platelets were untreated or treated with W7 (150 μM, final concentration) for 1 hour in the presence of EDTA, then platelets were lysed and immunoprecipitated with nonimmune mouse (NIM) IgG or anti-FcγRIIa mAb, IV.3. Immunoprecipitates were captured on protein A/G-Sepharose and analyzed by Western blotting with anti–FcγRIIa tail antiserum (top panel) or anti–calmodulin mAb (bottom). (D) Levels of calmodulin associated with FcγRIIa after treatment of washed platelets with W7, 14A2, or VM58 were assessed by immunoprecipitation using a polyclonal antibody against the FcγRIIa extracellular domain, followed by Western blot with an anti–calmodulin antibody. All Western blots were visualized with HRP-conjugated secondary antibodies and ECL. Vertical lines have been inserted to indicate a repositioned gel lane. All lanes within each figure came from the same experiment and the same gel/Western blot.