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 recen tly cloned B2 kinin receptor which belongs to the superfamily of recep tors with seven transmembrane domains. The molecular events which tran sduce the bradykinin signal on the post-receptor level are not underst ood in detail. We studied whether in human foreskin fibroblasts bradyk inin treatment induces tyrosine phosphorylation of cellular proteins. Using phosphotyrosine antibodies we detected a bradykinin-dependent ph osphorylation of a group of proteins of about 130 kDa and an additiona l signal around 70 kDa after starvation of cells. The effect evoked by 10 nM bradykinin was rapid (2 min) and it was partially reduced by th e B2-kinin-receptor antagonist Hoe 140 which was shown to be a weak in ducer of tyrosine phosphorylation. The bradykinin-mediated tyrosine ph osphorylation events were reproduced in human embryonal kidney 293 fib roblasts which were transiently transfected with the rat B2 kinin rece ptor, but they were not observed in untransfected 293 control cells. T hese data suggest that the B2 kinin-receptor subtype is involved. Upon fractionation of cells the 130-kDa protein group was recovered both h i the membrane and the cytosolic protein fraction. To assess the speci ficity of this bradykinin effect we stimulated human foreskin fibrobla sts 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 produc ed by bradykinin. Immunoprecipitation experiments with specific antibo dies against potential candidate proteins in the molecular-mass range around 130 kDa revealed positive results for the focal adhesion kinase FAK and the p130 Src substrate while negative results were obtained f or the GTPase-activating protein GAP, the phospholipase C-gamma 1, the Janus kinase JAK-1 and vinculin. The data suggest that the tyrosine p hosphorylation of FAK and the p130 Src substrate might be involved in the B2-kinin-receptor signalling cascade.