G-protein coupled receptor-mediated activation of PI 3-kinase in neutrophils.
Stimulation of the respiratory burst of neutrophil leukocytes with chemotactic agonists requires two concomitant signal transduction pathways. One is calcium dependent and leads to activation of phospholipase C, the other is calcium independent but sensitive to the fungal metabolite wortmannin, a specific inhibitor of phosphatidylinositide 3-kinase (PI 3-kinase). Two isoforms of PI 3-kinase have been characterized in neutrophils, the p85/p110 PI 3-kinase alpha and the p101/p120 PI 3-kinase gamma. The relative contribution of the two PI 3-kinases in mediating chemoattractant-stimulated superoxide production and exocytosis in neutrophils in unclear. Here, we report that the protein tyrosine kinase inhibitor genistein markedly attenuates chemoattractant-stimulated phosphatidylinositol (3,4,5)-trisphosphate (PIP3) formation in neutrophils. PI 3-kinase activity in untreated cells is bimodal showing a maximum production after 10-15 sec that protracts with a lower PIP3 formation for approximately 2 min and returns to basal levels after 2-3 min. Genistein at 100 microM strongly inhibits PIP3 elevation and the fMet-Leu-Phe-stimulated respiratory burst. The activity of purified PI 3-kinase, however, is not altered in the presence of genistein, suggesting that the genistein-sensitive intermediate is located between the G-protein-coupled receptor and PI 3-kinase. Expression of a dominant negative form of PI 3-kinase alpha in GM-1/CXCR1 cells, a promyelolocytic cell line transfected with the G-protein-coupled receptors CXCR1, considerably reduces IL-8-stimulated PIP3 formation. The present observations suggest that in phagocytes stimulated with agonists of G-protein-coupled receptors the bulk of PIP3 is generated by PI 3-kinase alpha, which is activated through a genistein-sensitive target, presumably a protein tyrosine kinase.
Theodor Kocher-Institute, University of Berne, Switzerland. Thelen@tki.unibe.ch
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D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D015703:Antigens, CD / Q000096:biosynthesis / Q000502:physiology*; D002460:Cell Line; D002634:Chemotaxis, Leukocyte / Q000187:drug effects / Q000502:physiology; D016384:Consensus Sequence; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000097:blood*; D019833:Genistein / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D016209:Interleukin-8 / Q000494:pharmacology; D007700:Kinetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000097:blood*; D018121:Receptors, Cytokine / Q000502:physiology; D018123:Receptors, Interleukin / Q000096:biosynthesis / Q000502:physiology*; D023062:Receptors, Interleukin-8A; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D016897:Respiratory Burst / Q000187:drug effects*; D017384:Sequence Deletion; D014162:Transfection; D000077191:Wortmannin; D018909:src Homology Domains
D000730:Androstadienes; D015703:Antigens, CD; D004791:Enzyme Inhibitors; D016209:Interleukin-8; D018121:Receptors, Cytokine; D018123:Receptors, Interleukin; D023062:Receptors, Interleukin-8A; D011993:Recombinant Fusion Proteins; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019833:Genistein; D019869:Phosphatidylinositol 3-Kinases; D019204:GTP-Binding Proteins; D000077191:Wortmannin