Regulation of muscarinic cholinergic receptor endocytosis by phosphatidylinositol 4-kinase β

574

Vol. 64, Nos. 6/7, 1999

Abstracts

40 MUSCARINIC RECEPTOR-MEDIATED CTYOSKELETON IS COUPLED THROUGH SMOOTH MUSCLE CELLS.

REORGANIZATION THE Gia 2 PROTEIN

CW Emala, H Togashi, D Shao, CA Hirshman. Dept. of Anesthesiology, College of Physicians and Surgeons, New York, New York

OF THE ACTIN IN HUMAN AIRWAY

Columbia University

Muscarinic receptor signaling pathways that couple to contraction of airway smooth muscle (ASM) are also associated with reorganization of the actin cytoskeleton. We have recently shown that a pertussis-sensitive G protein, the small G protein, rho, and a tyrosine kinase are intermediate proteins linking the activation of muscarinic receptors to reorganization of the actin cytoskeleton. The current study was designed to determine which heterotrimeric G protein couples muscarinic receptors to the reorganization of the actin cytoskeleton (measured by an increase in the F/G actin ratio) in ASM. Cultured human airway smooth muscle cells expressing the Mz muscarinic receptor were grown to confluence. Cells were exposed for 6 days to 10 UM antisense oligonucleotides designed to specifically bind to the mRNA encoding Gio2, Gia3, or Gqa. A randomly scrambled oligonucleotide served as a control. F/G actin ratios were measured following 5 min of carbachol exposure which we have previously shown to increase the F/G actin ratio indicative of actin reorganization. Cells in parallel wells were harvested for immunoblot analysis of G protein a subunit expression. Results of immunoblots showed that 6 days of oligonucleotide antisense treatment decreased protein expression of the respective G protein a subunit. Antisense depletion of the Gia2 protein, but not the Gp3, nor G,a protein blocked the carbachol-induced increase in the F/G actin ratio. These results show that the Gia2 protein couples muscarinic receptors to the reorganization of the actin cytoskeleton in airway smooth muscle.

41 REGULATION OF MUSCARINIC CHOLINERGIC RECEPTOR ENDOCYTOSIS BY PHOSPHATIDYLINOSITOL 4-KINASE B S. D. Sorensen, D. A. Linseman, E. L. McEwen, A. M. Heacock, and S. K. Fisher. Neuroscience Laboratory, Mental Health Research Institute and Department of Pharmacology, University of Michigan, Ann Arbor, MI 48104-1687, U.S.A. A role for phosphoinositides

in the endocytosis of muscarinic cholinergic receptors (mAChRs)

Pretreatment of SH-SYSY neuroblastoma cells with micromolar has been investigated. concentrations of either wortmannin (WT), LY-294002 or phenylarsine oxide (PAO), three chemically distinct agents known to inhibit phosphatidylinositol 4-kinase (PI4K), resulted in both an inhibition of agonist-induced endocytosis of mAChRs and a selective reduction in the 32PPAO-mediated inhibition of both receptor labeling of phosphatidylinositol 4-phosphate. endocytosis and phosphoinositide synthesis could be fully reversed by inclusion of the Although each of these inhibitors attenuate receptorbifunctional thiol, 2,3-dimercaptopropanol. mediated second messenger production, mAChR internalization was readily observed under conditions in which PLC activity is essentially abolished ([Ca2+] 75%. Determination subcellular fractions of SH-SYSY cells indicated that WT, LY-294002 and PA0 preferentially inhibited enzyme activity in endocytic and cytosolic fractions, a profile consistent with the subcellular distribution of the 110 kDa p-isoform of PI4K, as determined by western blot analysis. Activity of PI4KP present in immunoprecipitated cell lysates was inhibited >75% by These results indicate that the ongoing synthesis of inclusion of each of the three inhibitors. phosphoinositides is necessary for mAChR endocytosis and support a role for the PI4KP isoform in regulating the phosphoinositide pool required for the maintenance of receptor endocytosis. (Supported by NS 23831, MH 46252, and GM 07767.)