P2X4 and P2X6 receptors associate with VE-cadherin in human endothelial cells

We investigated the expression of P2X(4) and P2X(6) receptors on human umbilical vein endothelial cells (HUVECs) and found that both P2X receptor subtypes on plasma membranes are largely restricted to areas of cell-cell contact. Co-labelling experiments at the confocal and electron microscopy levels revealed that P2X(4) and P2X(6) receptors are strongly co-localised with the cell adhesion molecule VE-cadherin. The P2X(4) and P2X(6) receptors on plasma membranes at cellular junctions are rapidly (within 5 min) internalised specifically after decreasing extracellular [Ca2+]. Disruption of microfilaments, microtubules and integrin-mediated adhesion or stimulation of P2 receptors with ATP did not alter P2X(4) and P2X(6) receptor expression on HUVEC plasma membranes. Membraneous P2X(4) and P2X(6) receptors resisted extraction with Triton-X 100, whereas cytoplasmic P2X receptors were Triton-X 100 soluble. P2X(4) receptors, but not P2X(6) receptors, could be co-immunoprecipitated with VE-cadherin and vice versa. We conclude that P2X(4) and P2X(6) receptors are associated with VE-cadherin at HUVEC adherens junctions.