Signalling through phospholipase C interferes with clathrin-mediated endocytosis

Abstract

We investigated if phosphatidylinositol(4,5)bisphosphate (PtdIns(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 PtdIns(4,5)P 2-dependent. In the WKPT renal epithelial cell line, endocytosed insulin and β 2-glycoprotein I (β 2gpI) 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 μ 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 β 2gpI uptake. ATP stimulated phospholipase C activity, and also suppressed uptake of insulin, but not β 2gpI. This effect was reversed by the PLC inhibitor U-73122. In polarized cell cultures, insulin uptake was apical, whereas β 2gpI 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.