Premium
Cell surface UT‐A1 is sequestered in lipid rafts and phosphorylated and ubiquitinated in non‐raft membrane domains
Author(s) -
Feng Xiuyan,
Yang Yuan,
Fröhlich Otto,
Klein Janet D,
Sands Jeff M,
Chen Guangping
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.970.11
Subject(s) - lipid raft , microbiology and biotechnology , ubiquitin , ubiquitin ligase , raft , chemistry , transfection , forskolin , biology , biochemistry , signal transduction , in vitro , organic chemistry , copolymer , gene , polymer
We previously reported that UT‐A1 mainly targets to lipid raft pools in both a heterologous expression system (UT‐A1‐MDCK cells) and in native rat IMCD suspensions. In this study, we investigated the possible role of lipid rafts in UT‐A1 regulation. Disruption of cell membrane lipid rafts by methyl‐β ‐cyclodextrin in Xenopus oocytes significantly increased UT‐A1 urea transport activity. When stimulated with forskolin, the distribution of UT‐A1 in the cell membrane fraction was shifted from lipid raft to non‐lipid fractions in UT‐A1‐MDCK cells. We then examined whether the UT‐A1 that was moved away from the lipid fraction could represent an activated form. We metabolically labeled UT‐A1‐MDCK cells with 32P‐orthophosphorylate; then treated the cells with 10 μM forskolin for 10 min. High levels of UT‐A1 phosphorylation were found in non‐lipid rafts. Dowregulation of UT‐A1 is accompanied via ubiquitination by E3 ligase MDM2. We further investigated cell surface UT‐A1 degradation. UT‐A1 293 cells were transfected with pCW7‐His‐ubiquitin. Ubiquitinated UT‐A1 was found in non‐lipid rafts. Interestingly, UT‐A1 ubiquitin ligase MDM2 is only identified in the non‐lipid raft fraction, which is consistent with the predominance of UT‐A1 ubiquitination and degradation outside of rafts. These findings suggest a new mechanism for regulation of UT‐A1 at the cell surface. UT‐A1 urea transport activity regulation involves movement between lipid and non‐lipid populations. Functional UT‐A1 is translocated in non‐lipid factions. UT‐A1 in rafts has low clearance. Lipid rafts sequester UT‐A1 away from the degradation machinery located in non‐raft membrane domains (MDM2).