Signalling through phospholipase C interferes with clathrin-mediated endocytosis

We investigated if phosphafidylinositol(4,5)bisphosphate (Ptdlns(4,5)P(2)) hydrolysis by phospholipase C activation through cell surface receptors would interfere with clathrin-mediated endocytosis as recruitment of clathrin assembly proteins is Ptdlns(4,5)P(2)-dependent. In the WKPT renal epithelial cell line, endocytosed insulin and beta(2)-glycoprotein I (beta(2)gpI) 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 mu(2)-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 beta(2)gpI uptake. ATP stimulated phospholipase C activity, and also suppressed uptake of insulin, but not beta gpI. This effect was reversed by the PLC inhibitor U-73122. In polarized cell cultures, insulin uptake was apical, whereas beta(2)gpI 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. (c) 2006 Elsevier Inc. All rights reserved.