sepharose and pervanadate

Ligand binding to the platelet-derived growth factor (PDGF) beta-receptor leads to increased receptor tyrosine phosphorylation as a consequence of dimerization-induced activation of the intrinsic receptor tyrosine kinase activity. In this study we asked whether ligand-stimulated PDGF beta-receptor tyrosine phosphorylation, to some extent, also involved reduced susceptibility to tyrosine dephosphorylation. To investigate this possibility we compared the sensitivity of ligand-stimulated and non-stimulated forms of tyrosine-phosphorylated PDGF beta-receptors to dephosphorylation using various preparations containing protein-tyrosine phosphatase activity. Ligand-stimulated or unstimulated tyrosine-phosphorylated receptors were obtained after incubation of cells with pervanadate only or pervanadate, together with PDGF-BB, respectively. Dephosphorylation of receptors immobilized on wheat germ agglutinin-Sepharose, as well as of receptors in intact cell membranes, was investigated under conditions when rephosphorylation did not occur. As compared with unstimulated receptors the ligand-stimulated PDGF beta-receptors showed about 10-fold reduced sensitivity to dephosphorylation by cell membranes, a recombinant form of the catalytic domain of density-enhanced phosphatase-1, or recombinant protein-tyrosine phosphatase 1B. We conclude that ligand-stimulated forms of the PDGF beta-receptor display a reduced susceptibility to dephosphorylation. Our findings suggest a novel mechanism whereby ligand stimulation of PDGF beta-receptor, and possibly other tyrosine kinase receptors, leads to a net increase in receptor tyrosine phosphorylation.

Topics: Agglutinins; Animals; Catalytic Domain; Cell Membrane; Cells, Cultured; Dimerization; Endothelium, Vascular; Enzyme Inhibitors; Ligands; Phosphorylation; Precipitin Tests; Protein Binding; Protein Phosphatase 1; Protein Structure, Tertiary; Protein Tyrosine Phosphatases; Receptor, Platelet-Derived Growth Factor beta; Recombinant Proteins; Sepharose; Swine; Transfection; Tyrosine; Vanadates