Functional involvement of Annexin-2 in cAMP induced AQP2 trafficking.
Annexin-2 is required for the apical transport in epithelial cells. In this study, we investigated the involvement of annexin-2 in cAMP-induced aquaporin-2 (AQP2) translocation to the apical membrane in renal cells. We found that the cAMP-elevating agent forskolin increased annexin-2 abundance in the plasma membrane enriched fraction with a parallel decrease in the soluble fraction. Interestingly, forskolin stimulation resulted in annexin-2 enrichment in lipid rafts, suggesting that hormonal stimulation might be responsible for a new configuration of membrane interacting proteins involved in the fusion of AQP2 vesicles to the apical plasma membrane. To investigate the functional involvement of annexin-2 in AQP2 exocytosis, the fusion process between purified AQP2 membrane vesicles and plasma membranes was reconstructed in vitro and monitored by a fluorescence assay. An N-terminal peptide that comprises 14 residues of annexin-2 and that includes the binding site for the calcium binding protein p11 strongly inhibited the fusion process. Preincubation of cells with this annexin-2 peptide also failed to increase the osmotic water permeability in the presence of forskolin in intact cells. Altogether, these data demonstrate that annexin-2 is required for cAMP-induced AQP2 exocytosis in renal cells.
Tamma
Grazia
G
Department of General and Environmental Physiology, University of Bari, Via Amendola 165/A, Bari, Italy.
Procino
Giuseppe
G
Mola
Maria Grazia
MG
Svelto
Maria
M
Valenti
Giovanna
G
D017306:Annexin A2 / Q000502:physiology*; D051399:Aquaporin 2 / Q000187:drug effects / Q000378:metabolism*; D002462:Cell Membrane / Q000502:physiology; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D000242:Cyclic AMP / Q000494:pharmacology*; D003600:Cytosol / Q000378:metabolism; D018076:DNA, Complementary / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008561:Membrane Fusion / Q000187:drug effects; D021962:Membrane Microdomains / Q000378:metabolism; D009995:Osmosis; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism
C472822:ANXA2 protein, human; D017306:Annexin A2; D051399:Aquaporin 2; D018076:DNA, Complementary; D012313:RNA; D000242:Cyclic AMP