Caveolin-1 directly interacts with UT-A1 urea transporter: the role of caveolae/lipid rafts in UT-A1 regulation at the cell membrane

Abstract

The cell plasma membrane contains specialized microdomains called lipid rafts which contain high amounts of sphingolipids and cholesterol. Lipid rafts are involved in a number of membrane protein functions. The urea transporter UT-A1, located in the kidney inner medullary collecting duct (IMCD), is important for urine concentrating ability. In this study, we investigated the possible role of lipid rafts in UT-A1 membrane regulation. Using sucrose gradient cell fractionation, we demonstrated that UT-A1 is concentrated in the caveolae-rich fraction both in stably expressing UT-A1 HEK293 cells and in freshly isolated kidney IMCD suspensions. In these gradients, UT-A1 at the cell plasma membrane is codistributed with caveolin-1, a major component of caveolae. The colocalization of UT-A1 in lipid rafts/caveolae was further confirmed in isolated caveolae from UT-A1-HEK293 cells. The direct association of UT-A1 and caveolin-1 was identified by immunoprecipitation and GST pull-down assay. Examination of internalized UT-A1 in pEGFP-UT-A1 transfected HEK293 cells fluorescent overlap with labeled cholera toxin subunit B, a marker of the caveolae-mediated endocytosis pathway. Disruption of lipid rafts by methyl-beta-cyclodextrin or knocking down caveolin-1 by small-interference RNA resulted in UT-A1 cell membrane accumulation. Functionally, overexpression of caveolin-1 in oocytes decreased UT-A1 urea transport activity and UT-A1 cell surface expression. Our results indicate that lipid rafts/caveolae participate in UT-A1 membrane regulation and this effect is mediated via a direct interaction of caveolin-1 with UT-A1.

Fig. 1.

Cell membrane UT-A1 is concentrated in lipid raft microdomains and codistributed with caveolin-1. UT-A1-HEK293 cells (A) or freshly prepared rat kidney inner medullary collecting duct (IMCD) suspension (B) were lysed with 1% Triton X-100 and loaded on 5–40% sucrose gradients for lipid raft isolation. After 20-h centrifugation, equal volume fractions were collected from the top to bottom and Western blotted for UT-A1. Caveolin-1 was used as a positive control for lipid raft fractions. Clathrin adaptor protein μ2 was used as the indicator for nonlipid raft fractions. The same membrane (B) was stripped and immunoblotted for aquaporin-2 (AQP2). The experiments of A were replicated in 10 separate experiments. B was repeated in 4 separate cohorts of animals with similar results.

Fig. 2.

UT-A1 associates with caveolin and colocalizes with lipid rafts. A: caveolae were isolated from UT-A1-HEK293 cells and blotted with UT-A1 antibody. UT-A1 was detected in the isolated caveolae. B: UT-A1 was immunoprecipitated from the total lysate and the isolated caveolae. The immunoprecipites were blotted for caveolin-1. *Unknown nonspecific band. C: GST pull-down assay. GST-caveolin-1 was incubated with rat IM tissue lysates (2 rats). The pulled-down samples were analyzed with UT-A1 antibody. D: HEK293 cells were transfected with pEGFP-UT-A1 for 48 h. Cells were fixed in 4% paraformaldehyde, incubated with Alexa-594-conjugated cholera toxin B (CTB), and visualized with a confocal microscope. A, B, and D were repeated twice. C was repeated 3 times.

Fig. 3.

UT-A1 endocytosis mediated by caveolae. A, left: schematic diagram shows the FLAG tag inserted in the first large extracellular loop. Right: Western blot analysis of FLAG-UT-A1 expression in HEK293 cells. FLAG-UT-A1 in pcDNA3 was transiently transfected into cells for 2 days. Cell lysates were immunoblotted with UT-A1 COOH-terminus antibody or FLAG. The pcDNA3-UT-A1 and FLAG HSP70 were used as controls. B: FLAG-UT-A1-transfected HEK293 cells were incubated with FLAG antibody and Alexa-CTB for 45 min on ice. Cell endocytosis was induced by incubation with prewarmed complete cell medium at 37°C for 5 min and analyzed under confocal microscope as described in materials and methods. Arrows indicate sites of colocalization as determined by yellow color. The results are representative of 2 separate experiments.

Fig. 4.

Knock-down of caveolin-1 by RNA interference increases UT-A1 membrane expression. UT-A1-HEK293 cells were transfected with pSuppressor/scramble, dynamin, or caveolin-1 for 48 h. Cells were processed for biotinylation. Total cell lysates and biotinylated samples were separated on SDS-PAGE and probed with antibodies against the indicated proteins. The experiments were repeated 3 times with similar results.

Fig. 5.

Lipid raft disruption by methyl-β-cyclodextrin (MβCD) increases UT-A1 expression at the cell surface. Three days after cRNA microinjection, the oocytes were incubated with 5 mM MβCD for 2 h at room temperature and then processed for [¹⁴C]urea uptake assay (n = 5∼6 oocytes/time point; A) or cell surface UT-A1 expression by biotinylation (B). This experiment was repeated 2 additional times with similar results. **P < 0.01.

Fig. 6.

Overexpression of caveolin-1 reduces UT-A1 cell surface protein expression and urea transport activity. Oocytes were coinjected with cRNAs encoding UT-A1 (2 ng/cell), plus dynamin or caveolin-1 (5 ng/cell) for 3 days. A: urea transport activity was measured by [¹⁴C]urea flux (n = 5∼6 oocytes/time point). *P < 0.05; **P < 0.01. B: cell surface UT-A1 protein expression was measured by biotinylation. These results were reproduced 2 additional times.