Pool of ligand-bound platelet-derived growth factor beta-receptors remain activated and tyrosine phosphorylated after internalization.
We have examined the state of tyrosine phosphorylation of ligand-bound, internalized platelet-derived growth factor (PDGF) beta-receptors. Analysis by immunofluorescence staining of cells stimulated with PDGF-BB at 37 degrees C indicated colocalization of phosphotyrosine, PDGF beta-receptors, and PDGF-BB in endosome-like vesicles. Treatment of cells with an acidic buffer, which removed cell surface-bound PDGF-BB, led to a considerable decrease in phosphorylation and kinase activity of cell surface localized PDGF beta-receptors, but not of internalized receptors. Immunoprecipitations using antisera against phosphotyrosine and the PDGF beta-receptor from metabolically labeled cells showed that a major fraction of the tyrosine-phosphorylated pool of receptors were still accessible to the acid buffer treatment after 10 min of incubation of the cells at 37 degrees C. Under these conditions, about 20-25% of the total pool of tyrosine-phosphorylated receptors were intracellular, since they remained tyrosine phosphorylated after the acid buffer treatment. A considerable pool of tyrosine-phosphorylated, internalized receptors, after 10 min of incubation of the cells at 37 degrees C, could also be detected by immunoblotting analysis, using antisera against the PDGF beta-receptor and phosphotyrosine. Analysis by in vitro kinase assays of immunoprecipitated PDGF beta-receptors, obtained from PDGF-BB-stimulated cells different times after acid wash, showed that the internalized receptors retained kinase activity. These data suggest that a pool of internalized PDGF beta-receptors remain active and may participate in signalling a considerable time after internalization.
Sorkin
A
A
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Eriksson
A
A
Heldin
C H
CH
Westermark
B
B
Claesson-Welsh
L
L
D000818:Animals; D001011:Aorta / Q000166:cytology; D015153:Blotting, Western; D002460:Cell Line; D004705:Endocytosis / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D007536:Isomerism; D008024:Ligands; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D019000:Phosphotyrosine; D010982:Platelet-Derived Growth Factor / Q000378:metabolism / Q000494:pharmacology; D011233:Precipitin Tests; D017479:Receptors, Platelet-Derived Growth Factor / Q000032:analysis / Q000378:metabolism / Q000502:physiology*; D013552:Swine; D013997:Time Factors; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism*
D008024:Ligands; D010982:Platelet-Derived Growth Factor; D019000:Phosphotyrosine; D014443:Tyrosine; D017479:Receptors, Platelet-Derived Growth Factor