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

Abstract

Blood sinusoidal plasma membrane subfractions were isolated from normal mouse liver in the presence of the proteinase inhibitors PhMeSO2F [phenylmethanesulfonyl fluoride] and iodoacetamide. They were purified from smooth microsomal and Golgi vesicle contaminants. The phosphorylation reaction was studied at 33.degree. C, in the presence of 2 mM MnCl2. Addition of epidermal growth factor (EGF) to the preparations stimulated 32P incorporation from [.gamma.-32P]ATP or [y-32P]GTP essentially into one 170,000 MW protein. Some incorporation was observed in a minor 120,000-MW component which appears to be a degradation product of the 170,000-MW component was observed after 4 min of incubation at 33.degree. C. This dephosphorylation reaction was inhibited by addition of 5 mM p-nitrophenyl phosphate but not by addition of micromolar Zn2+, Be2+ or orthovanadate. The 170,000-MW protein specifically bound 125I-labeled EGF and appeared to be the hepatic EGF receptor. The EGF stimulatable kinase activity considerably enhances incorporation of 32P into tyrosine residues of the 170,000-MW EGF receptor at 33.degree. C. Tryptic peptide maps of the 32P-labeled 170,000-MW protein revealed a multiplicity of phosphorylated sites. Seven 32P-labeled phosphopeptides were observed after EGF stimulation, 3 of them being largely prominent. Tryptic peptide maps of the 170,000-MW protein after it was covalently linked to 125I-labeled EGF showed only one 125I-labeled peptide, the migration of which appeared different from that of 32P-labeled phosphopeptides. These findings were confirmed by V8 protease unidimensional peptide mapping of the 170,000-MW protein, labeled with 32P or 125I-EGF.