Formyl Met-Leu-Phe-Stimulated FPR1 Phosphorylation in Plate-Adherent Human Neutrophils: Enhanced Proteolysis but Lack of Inhibition by Platelet-Activating Factor

Figure 1

Cell-penetrating serine protease inhibitor diisopropylfluorophosphate blocks FPR1 cleavage in solid phase assay of FPR1 phosphorylation. Twenty-four wells of a 96-well microtiter plate were loaded with 0 to 50 × 10⁴ PMN. Cells had been treated with control (left half) or 3 mM DFP-containing buffer (right half) for 5 min as described in Section 2. The plated PMN were exposed to 1 μM fMLF in RPMI or vehicle control and allowed to incubate for 10 min at 37°C before quenching and washing the wells with 200 μL of ice-cold DPBS followed by extraction as described in Section 2. 40 μL of the final extract (2.7 × 10⁴, 5.3 × 10⁴, 8 × 10⁴, 10.7 × 10⁴, and 13.3 × 10⁴ cell equivalents/lane) was then loaded on two 28-well SDS-poly acrylamide gels, immunoblotted, and developed with NFPRa (upper) or NFPRb (lower) as described in Section 2. Relative molecular mass markers are labeled to the left of the blot. In the two leftmost sample lanes, 1 μM fMLF-exposed (S+) or unexposed (S−) suspension cells prepared as previously described using a comparable cell equivalent load were run as comparative controls. The broad band, of molecular weight ranges 50 to 70 kDa and marked by the two diagonally pointing, single-headed thin arrows, identifies FPR1. The thick, horizontal arrow labeled 25K-FPR1 upper and 25K-FPR1 shows the position of the putative 25 kDa fragments of FPR1. This species is clearly absent in DFP-treated PMN. The calculated Mr values of the significant bands are FPR1 = 61.4, FPR2 45.3, 39 K 39.7, 35 K 34.7, and 25K 25.6 kDa. Data shown is representative of 2 experiments.