Characterization of the epidermal‐growth‐factor‐dependent phosphorylation system from normal mouse‐liver sinusoidal plasma membranes

Blood sinusoidal plasma membrane subfractions were isolated from normal mouse liver in the presence of the proteinase inhibitors PhMeSO 2 F and iodoacetamide. They were purified from smooth microsomal and Golgi vesicle contaminants.1 The phosphorylation reaction was studied at 33°C, in the presence of 2 mM MnCl 2 . Addition of epidermal growth factor (EGF) to the preparations stimulated 32 P incorporation from [γ‐ 32 P]ATP or [γ‐ 32 P]GTP essentially into one 170000 M r protein. Some incorporation was observed in a minor 120000‐ M r component which appears to be a degradation product of the 170000‐ M r component. No EGF‐dependent phosphorylation of other membrane proteins or various exogenous proteins could be detected in vitro . 2 The dephosphorylation of the 170000‐ M r component was observed after 4 min of incubation at 33°C. This dephosphorylation reaction was inhibited by addition of 5 mM p ‐nitrophenyl phosphate but not by addition of micromolar Zn 2+ , Be 2+ or orthovanadate. 3 The 170000‐ M r protein specifically bound 125 I‐labeled EGF and thus appeared to be the hepatic EGF receptor. 4 The EGF stimulatable kinase activity considerably enhances incorporation of 32 P into tyrosine residues of the 170000‐ M r EGF receptor at 33°C. Tryptic peptide maps of the 32 P‐labeled 170000‐ M r protein revealed a multiplicity of phosphorylated sites. Seven 32 P‐labeled phosphopeptides were observed after EGF stimulation, three of them being largely prominent. Tryptic peptide maps of the 170000‐ M r protein after it was covalently linked to 125 I‐labeled EGF showed only one 125 I‐labeled peptide, the migration of which appeared different from that of 32 P‐labeled phosphopeptides. These findings were confirmed by V8 protease unidimensional peptide mapping of the 170000‐ M r protein, labeled with 32 P or 125 I‐EGF.