Bradykinin induces tyrosine phosphorylation in human foreskin fibroblasts and 293 cells transfected with rat B2 kinin receptor.
The intracellular effects of bradykinin are mediated through the recently cloned B2 kinin receptor which belongs to the superfamily of receptors with seven transmembrane domains. The molecular events which transduce the bradykinin signal on the post-receptor level are not understood in detail. We studied whether in human foreskin fibroblasts bradykinin treatment induces tyrosine phosphorylation of cellular proteins. Using phosphotyrosine antibodies we detected a bradykinin-dependent phosphorylation of a group of proteins of about 130 kDa and an additional signal around 70kDa after starvation of cells. The effect evoked by 10 nM bradykinin was rapid (2 min) and it was partially reduced by the B2-kinin-receptor antagonist Hoe 140 which was shown to be a weak inducer of tyrosine phosphorylation. The bradykinin-mediated tyrosine phosphorylation events were reproduced in human embryonal kidney 293 fibroblasts which were transiently transfected with the rat B2 kinin receptor, but they were not observed in untransfected 293 control cells. These data suggest that the B2 kinin-receptor subtype is involved. Upon fractionation of cells the 130kDa protein group was recovered both in the membrane and the cytosolic protein fraction. To assess the specificity of this bradykinin effect we stimulated human foreskin fibroblasts with epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I) and insulin. While IGF-I, insulin and EGF were almost ineffective, PDGF stimulated the tyrosine phosphorylation of 130-kDa bands with a similar pattern to that produced by bradykinin. Immunoprecipitation experiments with specific antibodies against potential candidate proteins in the molecular-mass range around 130kDa revealed positive results for the focal adhesion kinase FAK and the p130 Src substrate while negative results were obtained for the GTPase-activating protein GAP, the phospholipase C-gamma1, the Janus kinase JAK-1 and vinculin. The data suggest that the tyrosine phosphorylation of FAK and the pl30 Src substrate might be involved in the B2-kinin-receptor signalling cascade.
Tippmer
S
S
Institut für Diabetesforschung, München, Germany.
Bossenmaier
B
B
Häring
H
H
D000818:Animals; D001920:Bradykinin / Q000031:analogs & derivatives / Q000494:pharmacology*; D002460:Cell Line; D002478:Cells, Cultured; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007668:Kidney; D008297:Male; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D051381:Rats; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000096:biosynthesis / Q000187:drug effects / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism; D012867:Skin / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D007328:Insulin; D010750:Phosphoproteins; D010982:Platelet-Derived Growth Factor; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D004815:Epidermal Growth Factor; D007334:Insulin-Like Growth Factor I; C065679:icatibant; D001920:Bradykinin