A role for a wortmannin-sensitive phosphatidylinositol-4-kinase in the endocytosis of muscarinic cholinergic receptors.


A role for phosphoinositides in the endocytosis of muscarinic cholinergic receptors (mAChRs) has been investigated via inhibition of the activity of phosphatidylinositol-4-kinase (PI4K). Pretreatment of SH-SY5Y neuroblastoma cells with micromolar concentrations of wortmannin (WT), LY-294002, or phenylarsine oxide (PAO), three chemically distinct agents known to inhibit PI4K, resulted in both an inhibition of agonist-induced endocytosis of mAChRs and a selective reduction in the 32P-labeling of phosphatidylinositol-4-phosphate. PAO-mediated inhibition of both receptor endocytosis and phosphoinositide synthesis could be fully reversed by inclusion of the bifunctional thiol 2, 3-dimercaptopropanol. The requirement for phosphoinositide synthesis in mAChR endocytosis was independent of a role for these lipids in the maintenance of the cytoskeleton because disruption of the latter with cytochalasin D, ML-7, or colchicine failed to inhibit receptor internalization. Determination of PI4K activity in subcellular fractions of SH-SY5Y cells indicated that enzyme activity in fractions enriched in endocytic vesicles and cytosol was preferentially inhibited by WT, LY-294002, and PAO, a profile consistent with the subcellular distribution of the 110-kDa beta isoform of PI4K, as determined by Western blot analysis. Activity of PI4Kbeta present in immunoprecipitated cell lysates was inhibited >75% by inclusion of each of the three inhibitors. These results indicate that ongoing synthesis of phosphoinositides is necessary for mAChR endocytosis and that the activity of a WT-sensitive form of PI4K, such as PI4Kbeta, is required.

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@article{Sorensen1998ARF, title={A role for a wortmannin-sensitive phosphatidylinositol-4-kinase in the endocytosis of muscarinic cholinergic receptors.}, author={Scott D Sorensen and Daniel A Linseman and Edward L. McEwen and Anne M. Heacock and Stephen K . Fisher}, journal={Molecular pharmacology}, year={1998}, volume={53 5}, pages={827-36} }