Evidence for the platelet-derived growth factor-stimulated tyrosine phosphorylation of the platelet-derived growth factor receptor in vivo. Immunopurification using a monoclonal antibody to phosphotyrosine.
The addition of platelet-derived growth factor (PDGF) to intact BALB/c 3T3 cells results in the rapid (less than 1 min), dose-dependent phosphorylation of a number of proteins that could be isolated by a monoclonal antiphosphotyrosine antibody. The predominant tyrosinephosphorylated protein shared many characteristics with the PDGF receptor, including its molecular weight (170,000), isoelectric point (pI of about 4.2), its binding to DEAE-cellulose, and its pattern of binding to lectins. This 170-kDa protein, labeled with [35S] methionine, was substantially purified from PDGF-stimulated cells using the monoclonal anti-phosphotyrosine antibody but was not significantly immunopurified from unstimulated cells. At 37 degrees C, phosphorylation of the 170-kDa protein was maximal by 5-10 min of exposure to PDGF, and thereafter decreased rapidly. However, at 4 degrees C, the phosphorylation continued to increase after 3 h of exposure to PDGF. Subsequently, shifting the cells from 4 to 37 degrees C resulted in an additional rapid burst of tyrosine phosphorylation. Among the other PDGF-stimulated molecules, the most prominent and consistently observed was a cytosolic, acidic (pI of about 4.2), 74-kDa protein. These findings indicate that the action of PDGF in vivo is associated with the rapid and transient tyrosine phosphorylation of several membrane and cytosolic proteins; the most prominent of these proteins, isolated by monoclonal antibody to phosphotyrosine, is likely to be the PDGF receptor. The use of this antibody provides a new approach for purification of the PDGF receptor.
D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D007163:Immunosorbent Techniques; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D019000:Phosphotyrosine; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor; D013696:Temperature; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000276:immunology
D000911:Antibodies, Monoclonal; D010982:Platelet-Derived Growth Factor; D011956:Receptors, Cell Surface; D019000:Phosphotyrosine; D014443:Tyrosine; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor