Signalling through phospholipase C interferes with clathrin-mediated endocytosis

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Clathrin-mediated endocytosis is crucial for cellular uptake of various molecules.
• Phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2) is essential for recruiting clathrin assembly proteins.
• Activation of phospholipase C (PLC) hydrolyzes PtdIns(4,5)P2, potentially affecting endocytosis.

Purpose of the Study:

• To investigate whether PLC activation interferes with clathrin-mediated endocytosis.
• To determine the effect of extracellular ATP on the uptake of insulin and beta2-glycoprotein I (beta2gpI) in renal epithelial cells.
• To elucidate the mechanisms underlying the selective inhibition of endocytosis by ATP.

Main Methods:

• Utilized the WKPT renal epithelial cell line.
• Assessed ligand uptake (insulin and beta2gpI) using clathrin-dependent endocytosis markers (AP180-C).
• Employed RNA interference to silence the mu2-subunit of AP-2.
• Investigated the effect of extracellular ATP and PLC inhibitor (U-73122).
• Examined uptake in polarized cell cultures.

Main Results:

• Both insulin and beta2gpI uptake were clathrin-dependent but utilized distinct mechanisms.
• Extracellular ATP stimulated PLC activity and suppressed insulin uptake, but not beta2gpI uptake.
• ATP's inhibitory effect on insulin uptake was reversed by a PLC inhibitor.
• Insulin uptake occurred apically, while beta2gpI uptake occurred basolaterally.

Conclusions:

• Stimulation of apical G-protein-coupled P2Y receptors by ATP leads to PLC activation.
• This activation diminishes clathrin-mediated endocytosis of apical ligands like insulin.
• Basolateral endocytic mechanisms, such as beta2gpI uptake, remain unaffected.
• The study highlights differential regulation of endocytic pathways based on receptor localization and signaling.