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This Article Full Text (PDF) All Versions of this Article: ajpcell.00098.2009v2    most recent ajpcell.00098.2009v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Yui, N. Articles by Uchida, S. PubMed PubMed Citation Articles by Yui, N. Articles by Uchida, S.

Research Article

FAPP2 is required for aquaporin-2 apical sorting at trans-Golgi network in polarized MDCK cells

¹Tokyo Medical and Dental University

Submitted 4 March 2009 ; revised 9 September 2009 ; accepted in final form 28 September 2009

FAPP2 is an adaptor protein of phosphatidylinositol-4-phosphate (PI4P) and is involved in the transport of some apical cargos from the trans-Golgi network (TGN). To investigate whether the regulated apical transport of aquaporin 2 (AQP2) is involved in the FAPP2-dependent apical protein sorting machinery, we measured apical sorting of AQP2 in MDCK cells with or without FAPP2 knock-down. We established MDCK cell lines that stably express rat AQP2 without any tag sequence. Then, FAPP2-deficient stable cell lines were established from the AQP2-expressing cell lines by a retrovirus-mediated RNA interference system. In the established cell lines, AQP2 was detected in both apical and basolateral membranes. Forskolin increased only the apical localization of AQP2, which was not affected by basolateral treatment with 0.5% tannic acid, indicating that the forskolin-induced apical transport of AQP2 did not include the transcytotic pathway from basolateral to apical membranes but is a direct transport from TGN to the apical membranes. Using this cell line, we tested the effect of FAPP2 knock-down on the polarized AQP2 transport to plasma membranes and found that the forskolin-induced apical transport of AQP2 was completely abolished by FAPP2 knock-down. By contrast, basolateral localization of AQP2 was not affected by FAPP2 knock-down. AQP2 phosphorylation by forskolin was also impaired in FAPP2 knock-down MDCK cells. These results suggest that FAPP2 is necessary to generate AQP2-bearing vesicles at trans-Golgi that will undergo phosphorylation by PKA in subapical regions.

phosphatidylinositol; forskolin; shRNA; biotinylation assay