Co-immunoprecipitation of Sema4D and calmodulin from platelet lysates and identification of a calmodulin-binding sequence in Sema4D. (A) Platelets (5 × 108/mL) were treated with 10 μg/mL collagen for 30 seconds and lysed. Proteins were immunoprecipitated with anti-Sema4D N-terminal antibody (1E8G9) and immunoblotted with anti-calmodulin and an anti-Sema4D C-terminal antibody (mean ± standard error of the mean; **P < .01; n = 3). (B) Platelets were treated with 1.0 μΜ PMA for 30 seconds and immunoprecipitated with the anti-Sema4D N-terminal antibody. (C) An alignment of the putative calmodulin binding sequence (Arg762-Lys779) of Sema4D with known calmodulin-binding sequence of other platelet surface molecules GPVI (Arg294-Pro309), GPIbβ (Arg149-Arg164), and PECAM-1 (Arg599-Ala614). Identical amino acids or conserved substitutions are highlighted. (D) Helical wheel representation of the Sema4D sequence (Arg762-Lys779) and the GPVI sequence (Arg294-Pro309). Circles, hydrophilic residues; diamonds, hydrophobic residues; pentagons, potentially positively charged.
