TYROSINE PHOSPHORYLATION OF PHOSPHATIDYLINOSITOL 3-KINASE AND OF THE THROMBOXANE A(2) (TXA(2)) RECEPTOR BY THE TXA(2) MIMETIC I-BOP IN A7R5CELLS

Thromboxane A(2) (TXA(2)) interacts with its G-protein coupled recepto r, the TP receptor, to produce contraction and proliferation of vascul ar smooth muscle cells. We have shown previously that proliferation of primary cultures of vascular smooth muscle cells initiated by [1S-(1 alpha, 2 beta(5Z), 3 alpha(1E, 3R), 4 enyl)-7-oxabicyclo-[2.2.1]heptan -2yl]-5'-heptenoic acid (I-BOP), a stable TXA(2) mimetic, is mediated by activation of mitogen-activated protein (MAP) kinase. In the presen t study, we examined further the intracellular mediators involved in T XA(2) activation of vascular smooth muscle cells. Transient transfecti on of the cDNA for the TP receptor into A7r5 vascular smooth muscle ce lls resulted in expression of TP receptors with a receptor density, B- max, of 0.7 +/- 0.2 pmol/mg protein and a receptor affinity, K-d, of 0 .6 +/- 0.1 nM (N = 7). Mock transfected cells lacked significant recep tor expression. In TP receptor transfected cells, I-BOP increased the activation of MAP kinase 2-fold, stimulated tyrosine phosphorylation o f cellular proteins of relative molecular mass (M-r) of 140, 85, 60, 5 6, and 45 kDa, and increased the message for c-jun, a nuclear transcri ption factor involved in mitogenesis, 2.6-fold. Immunoblot analysis in dicated that the 85-kDa protein represented phosphoinositide 3-kinase (PI3-K), while the 60 kDa protein was the TP receptor. The activity of PI3-K was increased 3.5-fold by the addition of I-BOP (0.1 mu M). In summary, the present study demonstrated that stimulation of the TP rec eptor results in tyrosine phosphorylation of the receptor and of P13-K . (C) 1997 Elsevier Science Inc.