FIGURE 4.

Hypoosmolarity-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), AG1478 (1 μmol/liter) (A) or bumetanide (5 μmol/liter) (B) was added 30 min prior to the institution of hypoosmolarity (225 mosmol/liter). Liver samples were taken at the time points indicated, and phosphorylation of EGFR Tyr⁸⁴⁵, Tyr¹⁰¹⁵, 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 hypoosmolarity induced an RGD peptide- and PP-2-sensitive phosphorylation of EGFR residues Tyr⁸⁴⁵ and Tyr¹¹⁷³, whereas no phosphorylation at position Tyr¹⁰⁴⁵ became detectable within 60 min (A). AG1478 blunted insulin-induced Tyr¹¹⁷³ phosphorylation, whereas Tyr⁸⁴⁵ phosphorylation remained unchanged (A), suggestive of an EGFR Tyr⁸⁴⁵ transphosphorylation leading to EGFR Tyr¹¹⁷³ autophosphorylation as observed upon insulin perfusion (Fig. 1). In contrast to insulin-induced EGFR phosphorylation, bumetanide did not affect hypoosmotic-induced EGFR phosphorylation (B).