Perforin, PAF, and PAF receptor form a ternary complex.

PAF receptors were immunopurified from the biotinylated membrane of IFN-γ–treated K562 cells with the use of the specific anti–PAF-receptor mAb. Purified receptors were incubated with or without C18:0 [³H]PAF in the presence of Ca⁺⁺and Mg²⁺ and then exposed to YT2C2 cell cytoplasmic extracts for perforin-binding analysis. To confirm the presence of the 3 components (PAF receptor, PAF, perforin) in the same elution fraction, Western blot analysis using, respectively, anti-PAF receptor (A1), antiperforin (A2) mAbs, and [³H]PAF radioactivity detection (B) were performed. This procedure allowed simultaneous detection of PAF receptor at the molecular weight (mw) of 42-kd (A1, lane 2); perforin at the mw of 66 kd (A2, lane 2); and radioactivity due to the presence of [³H]PAF in the complex (B, column 2). In the absence of [³H]PAF (B, column 3), PAF receptor was still detected (A1, lane 3) but perforin failed to bind to the receptor (A2, lane 3). In the absence of PAF receptors (Jurkat cell membrane extracts, A1, lane 4), [³H]PAF (B, column 4) and perforin (A2, lane 4) failed to form the ternary complex. The YT2C2 cell line was used as negative control for PAF receptor expression (A1, lane 1), as positive control for perforin expression (A2, lane1), and as negative control for [³H]PAF binding (B, column 1).