In vivo phosphorylation and dephosphorylation of the platelet-derived growth factor receptor studied by immunoblot analysis with phosphotyrosine antibodies.
Antibodies against the synthetic hapten azobenzyl phosphonate which specifically crossreact with phosphotyrosine have been produced and used to detect the proteins phosphorylated in tyrosine following exposure of intact quiescent Swiss 3T3 fibroblasts to the platelet-derived growth factor (PDGF). Western blotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated proteins followed by decoration with phosphotyrosine antibodies and 125I-labeled protein A have been used. The major tyrosine-phosphorylated component was a 170 kDa protein. The following lines of evidence suggest that this protein is the PDGF receptor in its tyrosine-phosphorylated form: (a) both proteins have the same (170 kDa) molecular weight; (b) the phosphorylated 170 kDa protein was detectable only in cell lines bearing the PDGF receptor; (c) the phosphorylation of the 170 kDa protein required PDGF and was dose-dependent. Kinetic studies showed that the phosphorylation of the receptor was maximal after 5-10 min at 37 degrees C and was followed by a rapid decrement of the band. The loss of the 170 kDa component was not prevented by inhibitors of membrane internalization and of lysosomal proteinases, while it was inhibited by lowering the temperature to 5 degrees C. In PDGF-stimulated cells, phosphotyrosine antibodies detected also a minor 36 kDa component phosphorylated at tyrosine.
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Zippel
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Sturani
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Toschi
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Naldini
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Alberghina
P M
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Comoglio
D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002103:Cadaverine / Q000031:analogs & derivatives / Q000494:pharmacology; D002738:Chloroquine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D007158:Immunologic Techniques; D008744:Methylamines / Q000494:pharmacology; D051379:Mice; D008970:Molecular Weight; D010658:Phenylglyoxal / Q000494:pharmacology; D010766:Phosphorylation; D019000:Phosphotyrosine; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D008744:Methylamines; D010982:Platelet-Derived Growth Factor; D011956:Receptors, Cell Surface; D019000:Phosphotyrosine; D014443:Tyrosine; D002738:Chloroquine; D000255:Adenosine Triphosphate; C027451:methylamine; D017479:Receptors, Platelet-Derived Growth Factor; C008542:monodansylcadaverine; D002103:Cadaverine; D010658:Phenylglyoxal