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.
Sorensen
S D
SD
Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48104-1687, USA.
Linseman
D A
DA
McEwen
E L
EL
Heacock
A M
AM
Fisher
S K
SK
D019870:1-Phosphatidylinositol 4-Kinase / Q000037:antagonists & inhibitors*; D000730:Androstadienes / Q000494:pharmacology*; D001152:Arsenicals / Q000494:pharmacology; D002867:Chromones / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D004705:Endocytosis; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D018721:Muscarinic Agonists / Q000378:metabolism / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000096:biosynthesis; D011233:Precipitin Tests; D011485:Protein Binding; D011976:Receptors, Muscarinic / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D014407:Tumor Cells, Cultured; D000077191:Wortmannin
D000730:Androstadienes; D001152:Arsenicals; D002867:Chromones; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009025:Morpholines; D018721:Muscarinic Agonists; D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; C029341:oxophenylarsine; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D019870:1-Phosphatidylinositol 4-Kinase; D000077191:Wortmannin