PHOSPHATIDYLINOSITOL 3-KINASE ACTIVITY IS REQUIRED AT A POSTENDOCYTICSTEP 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 phosphatid ylinositol 3-kinase (PI 3-kinase) fail to concentrate in juxtanuclear vesicular structures after activation with PDGF. We have now identifie d the point in the endocytic pathway at which PI S-kinase binding site s are required. Receptor internalization from the plasma membrane, mea sured as the acquisition of acid resistance of prebound I-125-PDGF, wa s only slightly decreased in cells expressing a PDGF receptor mutant ( F5) lacking PI 3-kinase, GTPase-activating protein (GAP), phospholipas e C gamma, and Syp binding sites but not expressing mutants where any of these individual sites were restored nor expressing a mutant lackin g exclusively PI 3-kinase binding sites. In contrast, the extent of do wn-regulation of PDGF binding sites from the cell surface after prolon ged incubation with PDGF as well as the degradation of [S-35]methionin e-labeled receptor were markedly reduced in cells expressing the F5 mu tant, mutants restored in GAP, phospholipase C gamma, or Syp binding s ites 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 8-kinase activity with wortmann in caused a dramatic decrease in the rates of down-regulation and degr adation of wildtype receptors. These results suggest that PI 3-kinase binding sites are not required for internalization of PDGF receptor bu t are required to divert the PDGF receptor to a degradative pathway. F urthermore, the requirement for PI 3-kinase binding sites on the recep tor appears to be due to a requirement for PI 8-kinase catalytic activ ity.