Hope, H.R. and Pike, L.J. (1996) Phosphoinositides and Phosphoinositide-utilizing Enzymes in Detergent-insoluble Lipid Domains. Mol. Biol. Cell 7: 843-851.

Recent evidence has implicated caveolae/DIGs in various aspects of signal transduction, a process in which polyphosphoinositides play a central role. We therefore undertook a study to determine the distribution of phosphoinositides and the enzymes that utilize them in these detergent-insoluble domains. We report here that the polyphosphoinositide phosphatase, but not several other phosphoinositide-utilizing enzymes, is highly enriched in a low density, Triton-insoluble membrane fraction that contains caveolin. This fraction is also enriched in polyphosphoinositides, containing approximately one-fifth of the total cellular phosphatidylinositol (4,5)P2. Treatment of cells with the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), did not alter the distribution of polyphosphoinositides or the polyphosphoinositide phosphatase. However, PMA treatment did lead to a decrease in the mitogen-activated protein kinase and actin present in these domains. PMA also induced the recruitment of protein kinase to the caveolae/DIGs fraction. These findings suggest that polyphosphoinositides, the polyphosphoinositide phosphatase and protein kinase C play an important role in the structure or function of detergent-insoluble receptor exhibited both high and low affinity binding sites for 125I-EGF, the EGFR/RET chimera exhibited only low affinity binding of 125I-EGF. The chimera was able to internalize EGF more rapidly than the wild type EGF receptor and recycled to the cell surface at twice the rate of the EGF receptor. Pulse-chase experiments indicated that EGF stimulated the degradation of the wild type EGF receptor but had no effect on the rate of degradation of the EGFR/RET receptor. The combination of increased recycling and decreased degradation resulted in the relatively inefficient down-regulation of the EGFR/RET chimera. Incubation of cells expressing the wild type EGF receptor with phorbol 12-myristate 13-acetate led to a reduction in 125I-EGF binding and a loss in EGF-stimulated tyrosine phosphorylation. However, phorbol 12-myristate 13-acetate treatment had only a limited effect on EGF binding and EGF-stimulated tyrosine kinase activity in cells expressing EGFR/RET chimeras. These findings suggest that the ret tyrosine kinase is not regulated by many of the common mechanisms used to terminate signaling via growth factor receptors. Such persistent activation of the Ret tyrosine kinase may be relevant to the physiological function of Ret in cells that normally express this growth factor receptor.