title: Signalling through phospholipase C interferes with clathrin-mediated endocytosis.
abstract: We investigated if phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2) hydrolysis by phospholipase C activation through cell surface receptors would interfere with clathrin-mediated endocytosis as recruitment of clathrin assembly proteins is PtdIns(4,5)P2-dependent. In the WKPT renal epithelial cell line, endocytosed insulin and beta2-glycoprotein I (beta2gpI) were observed in separate compartments, although endocytosis of both ligands was clathrin-dependent as demonstrated by expression of the clathrin-binding C-terminal domain of AP180 (AP180-C). The two uptake mechanisms were different as only insulin uptake was reduced when the mu2-subunit of the adaptor complex AP-2 was silenced by RNA interference. ATP receptors are expressed at the apical surface of renal cells and, thus, we examined the effect of extracellular ATP on insulin and beta2gpI uptake. ATP stimulated phospholipase C activity, and also suppressed uptake of insulin, but not beta2gpI. This effect was reversed by the PLC inhibitor U-73122. In polarized cell cultures, insulin uptake was apical, whereas beta2gpI uptake was through the basolateral membrane, thus providing an explanation for selective inhibition of insulin endocytosis by ATP. Taken together, these results demonstrate that stimulation of apical G-protein-coupled P2Y receptors, which are coupled to phospholipase C activation diminishes clathrin-mediated endocytosis without interfering with basolateral endocytic mechanisms.
authors.affiliation: Department of Physiology, University College London, London WC1E 6JJ UK.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Carvou
authors.forename: Anthony G W
authors.initials: AG
authors.lastname: Norden
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Unwin
authors.forename: Shamshad
authors.initials: S
authors.lastname: Cockcroft
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002966:Clathrin / Q000502:physiology*; D004705:Endocytosis; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000502:physiology; D004963:Estrenes / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism; D034041:Monomeric Clathrin Assembly Proteins / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D015398:Signal Transduction; D050656:Transcription Factor AP-2 / Q000378:metabolism; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000502:physiology*; D019459:Urothelium / Q000166:cytology; D053482:beta 2-Glycoprotein I / Q000378:metabolism
chemical_list: D002966:Clathrin; D004963:Estrenes; D007328:Insulin; D034041:Monomeric Clathrin Assembly Proteins; D011760:Pyrrolidinones; D018048:Receptors, Purinergic P2; D050656:Transcription Factor AP-2; D053482:beta 2-Glycoprotein I; C439023:clathrin assembly protein AP180; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C068961:phosphatidylinositol 4,5-biphosphate kinase; D010738:Type C Phospholipases