BINDING AND INTERNALIZATION OF EXTRACELLULAR TYPE-I PHOSPHOLIPASE A(2) IN UTERINE STROMAL CELLS

The cellular uptake of extracellular type-I phospholipase A(2) (PLA(2) ) was investigated in rat uterine stromal cells (U-III) in culture, wh ich were found to express the high-affinity binding site for mammalian type-I PLA(2), with a measured K-D of 6.4 nM, a B-max of 0.1-1 pmol/m g of DNA at 4 degrees C, and a molecular mass of about 200 kDa. When U -III cells were treated with type-I PLA(2) at 37 degrees C, the ligand specifically associated with the cells increased, reaching a plateau after 90 min of incubation, whose level was about 5-fold higher than t hat measured if cells were maintained at 4 degrees C. We could determi ne that the PLA(2) was bound to plasma membrane receptors which were r esponsible for internalization of the ligand, and that the binding sit es were still suitable for binding at the level of plasma membrane dur ing U-III cell incubation at 37 degrees C. Proteolysis of internalized PLA(2) could be clearly detected only after 90 min of U-III cell incu bation with the ligand at 37 degrees C, and most of the intracellular PLA(2) consisted of the apparently intact 14 kDa enzyme. By cross-link ing studies, we found that most of the internalized PLA(2) was not ass ociated with the receptor, supporting the conclusion that in our exper imental system a single pool of membrane receptors for mammalian type- I PLA(2) undergoes cycles of ligand binding, intracellular transfer an d release of PLA(2), followed by restoration of binding sites on the p lasma membrane. We calculated that the rate of internalization of the ligand by one receptor molecule in U-III cells at 37 degrees C is abou t three molecules of type-I PLA(2) per h.