Inhibition of beta(2)-adrenergic and muscarinic cholinergic receptor endocytosis after depletion of phosphatidylinositol bisphosphate.
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).
Department of Pharmacology, University of Michigan, Ann Arbor, Michigan, USA.
S D
SD
Sorensen
D A
DA
Linseman
E L
EL
McEwen
A M
AM
Heacock
S K
SK
Fisher
D058672:Adrenergic beta-2 Receptor Antagonists; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology*; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D066298:In Vitro Techniques; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000037:antagonists & inhibitors*; D018129:Phosphatidylinositol Phosphates / Q000037:antagonists & inhibitors; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011869:Radioligand Assay; D051381:Rats; D011976:Receptors, Muscarinic / Q000187:drug effects*; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D014407:Tumor Cells, Cultured; D000077191:Wortmannin
D058672:Adrenergic beta-2 Receptor Antagonists; D000319:Adrenergic beta-Antagonists; D000730:Androstadienes; D004791:Enzyme Inhibitors; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011976:Receptors, Muscarinic; C037178:phosphatidylinositol 4-phosphate; D002118:Calcium; D000077191:Wortmannin