FIGURE 1.

Insulin-induced EGFR phosphorylation in perfused rat liver. Rat livers were perfused as described under “Experimental Procedures.” When indicated, RGD peptide (10 μmol/liter), PP-2 (250 nmol/liter), or AG1478 (1 μmol/liter) was added 30 min prior to the insulin institution (35 nmol/liter) to the perfusate to inhibit integrins, c-Src or EGFR tyrosine kinase activity, respectively. Liver samples were taken at the time points indicated, and phosphorylation of EGFR Tyr⁸⁴⁵, Tyr¹⁰⁴⁵, and Tyr¹¹⁷³ was analyzed by use of phospho-specific antibodies. Total EGFR served as loading control. Representative Western blots of three independent perfusion experiments are shown. Within 5 min insulin induced an RGD peptide- and PP-2-sensitive phosphorylation of EGFR residues Tyr⁸⁴⁵ and Tyr¹¹⁷³, whereas no phosphorylation at position Tyr¹⁰⁴⁵ is detectable within 60 min. AG1478 blunted insulin-induced Tyr¹¹⁷³ phosphorylation, whereas Tyr⁸⁴⁵ phosphorylation remained unchanged, suggestive of an EGFR Tyr⁸⁴⁵ transphosphorylation leading to EGFR Tyr¹¹⁷³ autophosphorylation.