Binding and internalization of extracellular type-I phospholipase A2 in uterine stromal cells.
The cellular uptake of extracellular type-I phospholipase A2 (PLA2) was investigated in rat uterine stromal cells (UIII) in culture, which were found to express the high-affinity binding site for mammalian type-I PLA2, with a measured KD of 6.4 nM, a Bmax of 0.1-1 pmol/mg of DNA at 4 degrees C, and a molecular mass of about 200 kDa. When UIII cells were treated with type-I PLA2 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 that measured if cells were maintained at 4 degrees C. We could determine that the PLA2 was bound to plasma membrane receptors which were responsible for internalization of the ligand, and that the binding sites were still suitable for binding at the level of plasma membrane during UIII cell incubation at 37 degrees C. Proteolysis of internalized PLA2 could be clearly detected only after 90 min of UIII cell incubation with the ligand at 37 degrees C, and most of the intracellular PLA2 consisted of the apparently intact 14 kDa enzyme. By cross-linking studies, we found that most of the internalized PLA2 was not associated with the receptor, supporting the conclusion that in our experimental system a single pool of membrane receptors for mammalian type-I PLA2 undergoes cycles of ligand binding, intracellular transfer and release of PLA2, followed by restoration of binding sites on the plasma membrane. We calculated that the rate of internalization of the ligand by one receptor molecule in UIII cells at 37 degrees C is about three molecules of type-I PLA2 per h.
Rossini
G P
GP
INSERM U. 352, Laboratoire de Physiologie Pharmacodynamie, Villeurbanne, France.
Fayard
J M
JM
Tessier
C
C
Laugier
C
C
D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active; D002478:Cells, Cultured; D003432:Cross-Linking Reagents; D005110:Extracellular Space / Q000201:enzymology; D005260:Female; D007700:Kinetics; D008024:Ligands; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism; D013552:Swine; D014599:Uterus / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D017819:Viperidae
D003432:Cross-Linking Reagents; D008024:Ligands; D011956:Receptors, Cell Surface; D010741:Phospholipases A; D054467:Phospholipases A2