Characterization of the molecular interaction between caveolin-1 and the P2X receptors 4 and 7 in E10 mouse lung alveolar epithelial cells

P2X4 and P2X7 receptors are abundantly expressed in alveolar epithelial cells, and are thought to play a role in regulating fluid haemostasis. Here, we analyzed the expression and localization of the P2X4R, and characterized the interaction between Cav-1 and both P2X4R and P2X7R in the mouse alveolar epithelial cell line E10. Using the biotinylation assay, we found that only glycosylated P2X4R is exposed at the cell surface. Triton X-100 solubility experiments and sucrose gradient centrifugation revealed that P2X4R was partially localized in Cav-1 rich membrane fractions. Cholesterol depletion with Mβ-CD displaced Cav-1 and P2X4R from the low-density to the high-density fractions. Suppression of Cav-1 protein expression using short hairpin RNAs resulted in a large reduction in P2X4R levels. Double immunofluorescence showed that P2X4R and Cav-1 partially colocalize in vitro. Using the GST pull-down assay, we showed that Cav-1 interacts in vitro with both P2X4R and P2X7R. Co-immunoprecipitation experiments confirmed the interaction between P2X7R and Cav-1. ATP stimulation increased the level of P2X4R in the lipid raft/caveolae fraction, whereas Cav-1 content remained constant. Our results support recent evidence that P2X receptors are present in both raft and non-raft compartments of the plasma membrane and thus exhibit variable ATP sensitivity.