Phosphatidylinositol 3-kinase activity is required at a postendocytic step in platelet-derived growth factor receptor trafficking.
We have previously reported that platelet-derived growth factor (PDGF) receptor mutants that lack high affinity binding sites for phosphatidylinositol 3-kinase (PI 3-kinase) fail to concentrate in juxtanuclear vesicular structures after activation with PDGF. We have now identified the point in the endocytic pathway at which PI 3-kinase binding sites are required. Receptor internalization from the plasma membrane, measured as the acquisition of acid resistance of prebound 125I-PDGF, was only slightly decreased in cells expressing a PDGF receptor mutant (F5) lacking PI 3-kinase, GTPase-activating protein (GAP), phospholipase C gamma, and Syp binding sites but not expressing mutants where any of these individual sites were restored nor expressing a mutant lacking exclusively PI 3-kinase binding sites. In contrast, the extent of down-regulation of PDGF binding sites from the cell surface after prolonged incubation with PDGF as well as the degradation of [35S]methionine-labeled receptor were markedly reduced in cells expressing the F5 mutant, mutants restored in GAP, phospholipase C gamma, or Syp binding sites or expressing the mutant exclusively lacking PI 3-kinase binding sites but not in cells expressing the mutant where PI 3-kinase binding sites were restored. Inhibition of PI 3-kinase activity with wortmannin caused a dramatic decrease in the rates of down-regulation and degradation of wild-type receptors. These results suggest that PI 3-kinase binding sites are not required for internalization of PDGF receptor but are required to divert the PDGF receptor to a degradative pathway. Furthermore, the requirement for PI 3-kinase binding sites on the receptor appears to be due to a requirement for PI 3-kinase catalytic activity.
Joly
M
M
Program in Molecular Medicine, University of Massachusetts Medical School, Worcester 01605, USA.
Kazlauskas
A
A
Corvera
S
S
D000730:Androstadienes / Q000494:pharmacology; D001665:Binding Sites; D001692:Biological Transport; D002460:Cell Line; D015536:Down-Regulation / Q000187:drug effects; D004705:Endocytosis; D006801:Humans; D006868:Hydrolysis; D019869:Phosphatidylinositol 3-Kinases; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D000077191:Wortmannin
D000730:Androstadienes; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D017479:Receptors, Platelet-Derived Growth Factor; D000077191:Wortmannin