Abstract
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.
Footnotes
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Send reprint requests to: Dr. Stephen K. Fisher, Neuroscience Laboratory, University of Michigan, 1103 E. Huron St., Ann Arbor, MI 48104-1687. E-mail: skfisher{at}umich.edu
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This work was supported by National Institutes of Health Grants NS23831 (S.K.F.) and MH46252 (S.K.F., A.M.H.). S.D.S. and D.A.L. were supported by National Institutes of Health Training Grant GM07767.
- Abbreviations:
- GPCR
- G protein-coupled receptor
- Oxo-M
- 2-butyn-1-ammonium,N,N,N-trimethyl-4-(2-oxo-1-pyrrolidinyl)iodide
- mAChR
- muscarinic acetylcholine receptor
- PEP
- priming of exocytosis proteins
- PLC
- phospholipase C
- PI
- phosphatidylinositol
- PIP
- phosphatidylinositol-4-phosphate
- PIP2
- phosphatidylinositol-4,5-bisphosphate
- PA
- phosphatidic acid
- PI4K
- phosphatidylinositol-4-kinase
- PI3K phosphatidylinositol-3-kinase
- PAO, phenylarsine oxide
- TCA
- trichloroacetic acid
- BAL
- 2,3-dimercaptopropanol (British anti-Lewisite)
- QNB
- quinuclidinyl benzilate
- NMS
- N-methylscopolamine
- WT
- wortmannin
- Received December 11, 1997.
- Accepted February 4, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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