Vol. 290, Issue 2, 603-610, August 1999

Inhibition of ₂-Adrenergic and Muscarinic Cholinergic Receptor Endocytosis after Depletion of Phosphatidylinositol Bisphosphate¹

Scott D. Sorensen, Daniel A. Linseman, Edward L. McEwen, Anne M. Heacock and Stephen K. Fisher

Neuroscience Laboratory, Mental Health Research Institute (E.L.M., A.M.H., S.K.F.); and Department of Pharmacology (S.D.S., D.A.L., S.K.F.), University of Michigan, Ann Arbor, Michigan

Recent evidence supporting a role for phosphoinositides in the endocytosis of phospholipase C-coupled receptors has prompted an investigation of whether there exists a similar requirement for the internalization of adenylyl cyclase-linked receptors. When 1321N1 astrocytoma cells, which possess both muscarinic cholinergic receptors (mAChRs) that couple to phospholipase C and -adrenergic receptors (₂-ARs) linked to adenylyl cyclase, were pretreated with wortmannin (WT) at a concentration known to inhibit phosphatidylinositol 4-kinase activity, the labeling of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PIP₂) was reduced. Stimulation of phosphoinositide breakdown by activation of mAChRs in WT-pretreated cells led to a further depletion of PIP₂. As previously demonstrated for SH-SY5Y neuroblastoma, inclusion of WT inhibited the endocytosis of mAChRs in 1321N1 cells by >85%. In contrast, the internalization of ₂-ARs was only partially (~30%) prevented. However, when the concentration of PIP₂ was further reduced by exposure of WT-pretreated 1321N1 cells to a muscarinic agonist, the endocytosis of ₂-ARs was substantially inhibited (>70%). Lower concentrations of WT (100 nM) that were sufficient to fully inhibit phosphatidylinositol 3-kinase activity had no effect on either phosphoinositide synthesis or receptor endocytosis. The results indicate that the agonist-induced endocytosis of an adenylyl cyclase-linked receptor such as the ₂-AR, like that of the phospholipase C-coupled mAChR, is dependent on the synthesis of phosphoinositides and, in particular, that of PIP₂.