10411568

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2

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 beta-adrenergic receptors (beta(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 (PIP(2)) was reduced. Stimulation of phosphoinositide breakdown by activation of mAChRs in WT-pretreated cells led to a further depletion of PIP(2). 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 beta(2)-ARs was only partially ( approximately 30%) prevented. However, when the concentration of PIP(2) was further reduced by exposure of WT-pretreated 1321N1 cells to a muscarinic agonist, the endocytosis of beta(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 beta(2)-AR, like that of the phospholipase C-coupled mAChR, is dependent on the synthesis of phosphoinositides and, in particular, that of PIP(2).

eng

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MEDLINE

0022-3565

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NLM

603-10

pubmed-meshheading:10411568-Adrenergic beta-2 Receptor Antagonists, pubmed-meshheading:10411568-Adrenergic beta-Antagonists, pubmed-meshheading:10411568-Androstadienes, pubmed-meshheading:10411568-Animals, pubmed-meshheading:10411568-Calcium, pubmed-meshheading:10411568-Calcium Signaling, pubmed-meshheading:10411568-Cytoplasm, pubmed-meshheading:10411568-Electrophoresis, Polyacrylamide Gel, pubmed-meshheading:10411568-Endocytosis, pubmed-meshheading:10411568-Enzyme Inhibitors, pubmed-meshheading:10411568-Humans, pubmed-meshheading:10411568-Immunoblotting, pubmed-meshheading:10411568-Phosphatidylinositol 3-Kinases, pubmed-meshheading:10411568-Phosphatidylinositol 4,5-Diphosphate, pubmed-meshheading:10411568-Phosphatidylinositol Phosphates, pubmed-meshheading:10411568-Radioligand Assay, pubmed-meshheading:10411568-Rats, pubmed-meshheading:10411568-Receptors, Muscarinic, pubmed-meshheading:10411568-Subcellular Fractions, pubmed-meshheading:10411568-Tumor Cells, Cultured

Inhibition of beta(2)-adrenergic and muscarinic cholinergic receptor endocytosis after depletion of phosphatidylinositol bisphosphate.

Journal Article, In Vitro, Research Support, U.S. Gov't, P.H.S.