Fig. 1. (A) A schematic picture of the ICAM-5 molecule with the amino acid sequence of the cytoplasmic domain and the peptides used in the ICAM-5--actinin interaction studies. The short peptides contain an additional C-terminal cysteine for coupling purposes. The peptides used in SPR analysis were biotinylated. PM, plasma membrane. (B) A GST-ICAM-5 cytodomain fusion protein binds -actinin. (a) A GST-cytoICAM-5 fusion protein immobilized on glutathione Sepharose beads was incubated with a Paju cell lysate, and interacting proteins characterized by western blotting, in this case an anti--actinin antibody. (b) Binding of purified -actinin to GST-cytoICAM-5, visualized by blotting. (C) Binding of purified -actinin to membrane proximal ICAM-5 cytoplasmic peptides. (a) Purified -actinin was incubated with the ICAM-5 cytodomain peptides (described in detail in Materials and Methods and in Fig. 1A) affinity matrices and the eluates were analyzed by SDS-PAGE together with purified -actinin, and immunoblotted with anti--actinin antibody. (b) Based on results shown above, the main -actinin-binding domain in the ICAM-5 cytodomain is located at residues 857-861 (box). (D) Binding of talin and filamin to ICAM-5_857-861 peptide. Paju cell lysates were incubated with the ICAM-5 peptide and the scrambled control peptide, and the eluates were run on SDS-PAGE and immunoblotted for talin (a) and filamin (b). No binding was observed. (E) Binding of -actinin to GGGKKGEY peptide as determined by SPR. Binding levels of -actinin at steady state of interaction with GGGKKGEY as response units (RU) after subtraction of control peptide sensorgrams from each GGGKKGEY binding sensorgram, respectively.