Characterization of the molecular interaction between caveolin-1 and the P2X receptors 4 and 7 in E10 mouse lung alveolar epithelial cells.
P2X(4) and P2X(7) 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 P2X(4)R, and characterized the interaction between Cav-1 and both P2X(4)R and P2X(7)R in the mouse alveolar epithelial cell line E10. Using the biotinylation assay, we found that only glycosylated P2X(4)R is exposed at the cell surface. Triton X-100 solubility experiments and sucrose gradient centrifugation revealed that P2X(4)R was partially localized in Cav-1 rich membrane fractions. Cholesterol depletion with Mbeta-CD displaced Cav-1 and P2X(4)R 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 P2X(4)R levels. Double immunofluorescence showed that P2X(4)R and Cav-1 partially colocalize in vitro. Using the GST pull-down assay, we showed that Cav-1 interacts in vitro with both P2X(4)R and P2X(7)R. Co-immunoprecipitation experiments confirmed the interaction between P2X(7)R and Cav-1. ATP stimulation increased the level of P2X(4)R 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.
Barth
K
K
Institute of Anatomy, Medical Faculty "Carl Gustav Carus", Dresden University of Technology, Fiedlerstr. 42, D-01307 Dresden, Germany.
Weinhold
K
K
Guenther
A
A
Linge
A
A
Gereke
M
M
Kasper
M
M
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism; D051242:Caveolin 1 / Q000378:metabolism*; D002460:Cell Line; D002784:Cholesterol / Q000172:deficiency; D015536:Down-Regulation / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006031:Glycosylation / Q000187:drug effects; D008168:Lung / Q000166:cytology*; D021962:Membrane Microdomains / Q000378:metabolism; D051379:Mice; D011485:Protein Binding / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011650:Pulmonary Alveoli / Q000166:cytology* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058848:Receptors, Purinergic P2X4; D058486:Receptors, Purinergic P2X7; D047392:beta-Cyclodextrins / Q000494:pharmacology
D051242:Caveolin 1; C548227:P2rx4 protein, mouse; C546469:P2rx7 protein, mouse; D034741:RNA, Small Interfering; D018048:Receptors, Purinergic P2; D058848:Receptors, Purinergic P2X4; D058486:Receptors, Purinergic P2X7; D047392:beta-Cyclodextrins; D000255:Adenosine Triphosphate; D002784:Cholesterol; C031215:betadex