Receptor heterodimerization: essential mechanism for platelet-derived growth factor-induced epidermal growth factor receptor transactivation

Abstract

Previous studies showed that the epidermal growth factor receptor (EGFR) can be transactivated by platelet-derived growth factor (PDGF) stimulation and that EGFR transactivation is required for PDGF-stimulated cell migration. To investigate the mechanism for cross talk between the PDGF beta receptor (PDGFbetaR) and the EGFR, we stimulated rat aortic vascular smooth muscle cells (VSMC) with 20 ng of PDGF/ml. Transactivation of the EGFR, defined by receptor tyrosine phosphorylation, occurred with the same time course as PDGFbetaR activation. Basal formation of PDGFbetaR-EGFR heterodimers was shown by coimmunoprecipitation studies, and interestingly, disruption of this receptor heterodimer abolished EGFR transactivation. Breakdown of the heterodimer was observed when VSMC were pretreated with antioxidants or with a Src family kinase inhibitor. Disruption of heterodimers decreased ERK1 and ERK2 activation by PDGF. Although PDGF-induced PDGFbetaR activation was abolished after pretreatment with 1 microM AG1295 (a specific PDGF receptor kinase inhibitor), EGFR transactivation was still observed, indicating that PDGFbetaR kinase activity is not required. In conclusion, our data demonstrate that the PDGFbetaR and the EGFR form PDGFbetaR-EGFR heterodimers basally, and we suggest that heterodimers represent a novel signaling complex which plays an important role in PDGF signal transduction.

FIG. 1

PDGF transactivates EGFR. Serum-starved VSMC were stimulated with 20 ng of PDGF-BB/ml for the times indicated. (A) Cell lysates were immunoprecipitated with anti-EGFR and immunoblotted with antiphosphotyrosine (4G10) (upper panel) and reprobed with anti-EGFR antibody (lower panel). The EGFR is shown as a single band at 170 kDa. (B) Relative phosphorylation of PDGFβR and EGFR. (C) The PDGFβR was coimmunoprecipitated with anti-EGFR. The PDGFβR and the EGFR were identified at 180 and 170 kDa, respectively. A band shown at 165 kDa (lanes 5 to 8) is nonspecific since it is not identified by 4G10. (D) Coprecipitation of EGFR and PDGFβR detected by diverse antibodies. Unstimulated cell lysates were immunoprecipitated with the anti-PDGFβR C-terminal domain (lane 1), the anti-PDGFβR kinase domain (lane 2), the anti-EGFR C-terminal domain (lane 3), and the anti-EGFR cytoplasmic domain (lane 4). Normal sheep IgG was used as a negative control for anti-EGFR (lane 5). Immunoblotting was performed with the anti-PDGFβR kinase domain. (E) Coprecipitation of PDGFβR by EGR antibody. Serum-starved VSMC were stimulated with 20 ng of PDGF-BB/ml for 5 min and treated with a cross-linker as described in Materials and Methods. Cell lysates were immunoprecipitated with the anti-EGFR C-terminal domain (lane 1), the anti-EGFR cytoplasmic domain (lane 2), the anti-PDGFβR C-terminal domain (lane 3), and the anti-PDGFβR kinase domain (lane 4). Normal rabbit IgG was used as a negative control for anti-PDGFβR. Immunoblotting was performed with the anti-EGFR C-terminal domain. (F) Serum-starved A431 cells were stimulated with 20 ng of PDGF-BB/ml or 10 ng of EGF/ml for 5 min. IP, immunoprecipitation, IB, immunoblotted.

FIG. 2

EGFR phosphorylation by pervanadate. Serum-starved VSMC were treated with 100 μM pervanadate for the times indicated. Cell lysates were immunoprecipitated with anti-EGFR and analyzed by immunoblotting, probed with 4G10 (upper panel), and reprobed with anti-EGFR (middle panel) and anti-PDGFβR (lower panel). IP, immuno precipitated; IB, immunoblotted.

FIG. 3

PDGF-induced EGFR transactivation after pretreatment with AG1295 or AG1478. Serum-starved VSMC were stimulated with 20 ng of PDGF-BB/ml for 5 min after preincubation with 10 μM AG1295 or 10 μM AG1478 for 30 min. Cell lysates were immunoprecipitated with anti-PDGFβR (A) or anti-EGFR (B) and analyzed by immunoblotting with 4G10 (upper panel) or anti-PDGFβR and anti-EGFR (lower panel). The PDGFβR and the EGFR were identified at 180 and 170 kDa, respectively. IP, immunoprecipitated; IB, immunoblotted.

FIG. 4

Effect of antioxidants on EGFR transactivation. Serum-starved VSMC were stimulated with 20 ng of PDGF-BB/ml for 5 min after pretreatment with 10 mM NAC or 10 mM Tiron for 60 min. (A) Cell lysates were immunoprecipitated with anti-EGFR and analyzed by immunoblotting, probed with 4G10 (upper panel), and reprobed with anti-EGFR (middle panel) or anti-PDGFβR (lower panel). IP, immunoprecipitated. IB, immunoblotted. (B) Relative tyrosine phosphorylation level of EGFR. Both NAC and Tiron significantly inhibited EGFR transactivation by PDGF. ∗, P < 0.001. (C) PDGFβR bound to EGFR. PDGFβR coimmunoprecipitated with EGFR significantly decreased after antioxidant treatment. ∗, P < 0.001.

FIG. 5

Effect of Src family kinase inhibitor on EGFR transactivation. Serum-starved VSMC were pretreated with 1 μM PP2 for 30 min, followed by stimulation with 20 ng of PDGF-BB/mi or 10 ng of EGF/ml for 5 min. (A) Cell lysates were immunoprecipitated with anti-PDGFβR (left panel) or anti-EGFR (right panel) and analyzed by immunoblotting with 4G10 as the probe. (B) Cell lysates were immunoblotted with anti-phospho-Src (Y416) and reprobed with anti-Src. (C) Cell lysates were immunoprecipitated with anti-EGFR, analyzed by immunoblotting with 4G10 as the probe (upper panel), and reprobed with anti-EGFR (middle panel) or anti-PDGFβR (lower panel) antibody. IP, immunoprecipitated. IB, immunoblotted. (D) Relative tyrosine phosphorylation level of EGFR. PP2 significantly inhibited EGFR transactivation by PDGF. †, P < 0.005. (E) PDGFβR bound to EGFR. The PDGFβR coimmunoprecipitated with the EGFR significantly decreased after antioxidant treatment. †, P < 0.005.

FIG. 6

Effect of receptor kinase inhibitors and antioxidants on ERK1/2 activity. Serum-starved VSMC were stimulated with 20 ng of PDGF-BB/ml after pretreatment with 1 μM AG1295 or 10 μM AG1478 for 30 min (A) or with 10 mM NAC or Tiron for 60 min or 1 μM PP2 for 30 min (B). Twenty micrograms of cell lysate was loaded onto each lane and analyzed by immunoblotting with anti-phospho-ERK1/2 antibody as the probe. Lower panels show the relative phosphorylation levels of ERK1/2. Each agent significantly inhibited ERK1/2 phosphorylation induced by PDGF stimulation. ∗, P < 0.001; †, P < 0.005; ‡, P < 0.01.

FIG. 7

Model of basal PDGFβR-EGFR heterodimer formation and signal transduction. PDGFβR and EGFR form a receptor complex (heterodimer) under basal cell conditions. The formation of the receptor complex may provide a scaffold for other molecules required for the transactivation. The interaction between the two receptors is regulated by ROS and Src family kinases. Note that we believe that the PDGFR dimers are physically closer than the EGFR dimers in three dimensions.