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

Abstract

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 p85alpha mutant lacking the p110-binding domain (Deltap85), 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 AT1 (Ang II 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-beta detected in VSMCs with an antibody against its cytoplasmic domain. Stimulation of VSMCs with Ang II increased tyrosine-phosphorylation of p70 PDGFR-beta at Tyr751 and Tyr1021 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 AT1 receptor antagonist, candesartan (CV 11974) and was partially inhibited by PP2 {4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine}, an Src family kinase inhibitor. Our result suggests that the p70 PDGFR-beta functions as an adapter that recruits PI3K to the membrane upon AT1 receptor stimulation.

Figure 1. Role of PI3K on Ang II-induced PAI-1 mRNA expression

Northern blot analysis shows the effect of LY294002 on Ang II-stimulated PAI-1 mRNA expression. Cells were pre-treated for 15 min with LY294002 at the concentrations indicated and stimulated with 100 nM Ang II for 3 h. (A) Northern blot analysis shows the effect of adenoviral transfection with Δp85 and β-galactosidase on Ang II-stimulated PAI-1 mRNA expression. (B) Representative Northern blots and a bar graph show quantification from three independent experiments (means±S.E.M.) with PAI-1. Significant effects on different treatment groups when compared with Ang II-stimulation alone or a control are indicated as *, P<0.05 and ***, P<0.001. 36B4, acidic ribosomal phosphoprotein.

Figure 2. Effects of PI3K and Src-kinase-inhibition on Ang II-induced PAI-1 protein expression

Western blot analysis and a bar graph show quantification of the effect of LY294002 on Ang II-stimulated PAI-1 protein expression. Cells were pre-treated with LY294002, stimulated with Ang II for 18 h, and PAI-1 level in the culture medium was measured. (A) Western blot analysis and a bar graph show quantification of the effect of the Src kinase inhibitor, PP2, on Ang II-stimulated PAI-1 protein expression. Cells were pre-treated with PP2, stimulated with Ang II for 18 h, and PAI-1 levels in the culture medium were measured. (B) Values are expressed as the fold-change compared with unstimulated or treated controls. Significant effects on different treatment groups when compared with Ang II stimulation alone or a control are indicated as *P<0.05; **P<0.01 and ***P<0.001. Values are expressed as the fold change compared with unstimulated or treated controls.

Figure 3. Identification of Ang II-stimulated p85 binding proteins

(A) VSMCs were stimulated with 100 nM Ang II for 5 min. Cell lysates were precipitated with an anti-(PI3K p85) antibody or the GST fusion protein containing the N-terminal SH2 domain of p85 and were subsequently Western blotted with an anti-p-Tyr antibody. Representative immunoblots (IB) and a bar graph show quantification of the tyrosine phosphorylation of a 70 kDa band are shown. The results are expressed as the fold change in stimulation over the basal level. Data shown are the means±S.E.M. for at least 3 independent experiments. *P<0.05; ***P<0.001. (B) PI3K-binding proteins were isolated from cell lysates of Ang II-simulated VSMCs and were stained with Coomassie Blue. The segment of the gel at 70 kDa was analysed by LC-MS/MS and the proteins identified are shown. (C) Schematic diagram shows the peptide coverage of the PDGFR-β identified by MS in the p70 kDa band. The locations of the peptide sequences identified in the p70 kDa band are indicated. The locations of peptides identified for the PDGFR-β isolated from the 190 kDa band are indicated as white bars. The phosphorylated tyrosine on p70 PDGFR-β, identified by LC-MS/MS, is indicated by (^−p). (D) MS/MS spectra of the Tyr⁷⁵¹-phosphorylated peptide (DESVDY-pVPMLDMK) of PDGFRβ, isolated from the 70 kDa band shown in Figure 3(B). Fragmentation y⁺ ions and b⁺ ions are labelled.

Figure 4. Ang II-stimulated p85-binding and tyrosine phosphorylation in a truncated PDGFR-β mutant

VSMCs were stimulated with Ang II. The resulting lysates were precipitated with GST–p85 fusion protein (A) or an anti-(PI3K p85) antibody (B) and immunoblotted (IB) with antibodies against the PDGFR-β C-terminal domain, cortactin, PTP or p-PDGFR-β (p-Tyr⁷⁵¹), as indicated. Representative immunoblots and a bar graph show quantification of p70 PDGFR-β phosphorylation (values are expressed as the fold change compared with unstimulated or treated controls; means±S.E.M.) for at least three independent experiments. Significant differences are indicated as *P<0.05.

Figure 5. Effect of Ang II on PDGFR-β phosphorylation

VSMCs were stimulated with 100 nM Ang II for 5 min and cell lysates were immunoblotted (IB) with an anti-PDGFR-β antibody, and anti-PDGFR-β phosphospecific antibodies against p-Tyr¹⁰²¹ and p-Tyr⁷⁵¹. Results are expressed as the fold change in stimulation (means±S.E.M) compared with unstimulated cells. Bar graphs show the results for at least three independent experiments. ***P<0.001; stimulated versus unstimulated cells.

Figure 6. Time course and effect of AT₁ receptor antagonism on Ang II-stimulated phosphorylation of p70 PDGFR-β

VSMCs were stimulated with Ang II for the indicated times (A). Cells were pre-treated with candesartan (CV 11974) for 30 min followed by stimulation with Ang II (100 nM) for 5 min (B). Immunoblotting was performed with anti-phospho-PDGFR-β (p-Tyr¹⁰²¹) antibodies. Results are expressed as the fold change in stimulation (means±S.E.M) compared with unstimulated cells. Bar graphs and immunoblots show the results for at least three independent experiments. Significant differences are indicated as ***P<0.001; **P<0.01; *P<0.05 for stimulated versus unstimulated cells.

Figure 7. Inhibition of p70 PDGFR-β phosphorylation

VSMCs were pre-treated with either AG1296 (A) or PP2 (B) for 15 min, followed by a 5 min stimulation with Ang II or PDGF, as indicated. Immunoblotting was performed with phospho-PDGFR-β (p-Tyr¹⁰²¹) antibodies. Results are expressed as fold change in stimulation (means±S.E.M.) compared with unstimulated cells. Bar graphs show the results for at least three independent experiments. Significant differences are indicated as *P<0.05 and **P<0.01.