RT Journal Article
SR Electronic
T1 A Role for a Wortmannin-Sensitive Phosphatidylinositol-4-Kinase in the Endocytosis of Muscarinic Cholinergic Receptors
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 827
OP 836
VO 53
IS 5
A1 Scott D. Sorensen
A1 Daniel A. Linseman
A1 Edward L. McEwen
A1 Anne M. Heacock
A1 Stephen K. Fisher
YR 1998
UL http://molpharm.aspetjournals.org/content/53/5/827.abstract
AB 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 β isoform of PI4K, as determined by Western blot analysis. Activity of PI4Kβ 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 PI4Kβ, is required. The American Society for Pharmacology and Experimental Therapeutics