Angiotensin II stimulates phosphorylation of an ectodomain-truncated platelet-derived growth factor receptor-β and its binding to class IA PI3K in vascular smooth muscle cells

PI3K (phosphoinositide 3-kinase) activity is involved in Ang (angiotensin) II-stimulated VSMC (vascular smooth muscle cell) growth and hypertrophy. In the present study, we demonstrate that the inhibition of PI3K in VSMCs by expression of a dominant-negative p85 alpha mutant lacking the p110-binding domain (Delta p85), or by treatment of cells with LY294002, inhibited Ang II-stimulated PAI-1 (plasminogen activator inhibitor-1) mRNA expression. Using a GST (glutathione S-transferase) fusion protein containing the p85 N-terminal SH2 (Src homology 2) domain as 'bait' followed by MS/MS (tandem MS), we identified a 70 kDa fragment of the p70 PDGFR-beta (platelet-derived growth factor receptor-beta) as a signalling adapter that is phosphorylated and recruits the p85 subunit of PI3K after Ang II stimulation of AT, (Ang 11 subtype 1) receptors on VSMCs. This fragment of the PDGFR-beta, which has a truncation of its extracellular domain, accounted for approx. 15% of the total PDGFR-detected in VSMCs with an antibody against its cytoplasmic domain. Stimulation of VSMCs with Ang 11 increased tyrosine-phosphorylation of p70 PDGFR-beta at Tyr(751) and Tyr(1021) and increased its binding to p85. PDGF also induced phosphorylation of p70 PDGFR-beta, a response inhibited by the PDGF tyrosine kinase selective inhibitor, AG1296. By contrast, Ang II-induced phosphorylation of the 70 kDa receptor was not affected by AG1296. Ang II-stimulated phosphorylation of the p70 PDGFR-beta was blocked by the AT, receptor antagonist, candesartan (CV 11974) and was partially inhibited by PP2 {4-amino-5(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]primidine}, an Src family kinase inhibitor. Our result suggests that the p70 PDGFR-beta functions as an adapter that recruits PI3K to the membrane upon AT(1) receptor stimulation.