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Pool of ligand‐bound platelet‐derived growth factor β‐receptors remain activated and tyrosine phosphorylated after internalization
Author(s) -
Sorkin Alexander,
Eriksson Anders,
Heldin CarlHenrik,
Westermark Bengt,
ClaessonWelsh Lena
Publication year - 1993
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041560221
Subject(s) - internalization , phosphorylation , microbiology and biotechnology , receptor , tyrosine phosphorylation , ligand (biochemistry) , platelet derived growth factor receptor , tyrosine , receptor tyrosine kinase , chemistry , growth factor , biology , biochemistry
We have examined the state of tyrosine phosphorylation of ligand‐bound, internalized platelet‐derived growth factor (PDGF) β‐receptors. Analysis by immunofluorescence staining of cells stimulated with PDGF‐BB at 37 0 C indicated colocalization of phosphotyrosine, PDGF β‐receptors, and PDGF‐BB in endosome‐like vesicles. Treatment of cells with an acidic buffer, which removed cell surfacebound PDGF‐BB, led to a considerable decrease in phosphorylation and kinase activity of cell surface localized PDGF β‐receptors, but not of internalized receptors. Immunoprecipitations using antisera against phosphotyrosine and the PDGF β‐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 0 C. Under these conditions, about 20‐25% of the total pool of tyrosine‐phosphorylated, receptors were intratcellular, 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 0 C, could also be detected by immunoblotting analysis, using antisera against the PDGF β‐receptor and phosphotyrosine. Analysis by in vitro kinase assays of immunoprecipitated PDGF β‐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 β‐receptors remain active and may participate in signalling a considerable time after internalization. © 1993 Wiley‐Liss, Inc.