Pietrement Christine, Da Silva Nicolas, Silberstein Claudia, James Marianne, Marsolais Mireille, Van Hoek Alfred, Brown Dennis, Pastor-Soler Nuria, Ameen Nadia, Laprade Raynald, Ramesh Vijaya, Breton Sylvie
Center for Systems Biology, Program in Membrane Biology/Nephrology Division, Simches Research Center, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA.
J Biol Chem. 2008 Feb 1;283(5):2986-96. doi: 10.1074/jbc.M704678200. Epub 2007 Nov 30.
Water and solute transport across the plasma membrane of cells is a crucial biological function that is mediated mainly by aquaporins and aquaglyceroporins. The regulation of these membrane proteins is still incompletely understood. Using the male reproductive tract as a model system in which water and glycerol transport are critical for the establishment of fertility, we now report a novel pathway for the regulation of aquaporin 9 (AQP9) permeability. AQP9 is the major aquaglyceroporin of the epididymis, liver, and peripheral leukocytes, and its COOH-terminal portion contains a putative PDZ binding motif (SVIM). Here we show that NHERF1, cystic fibrosis transmembrane conductance regulator (CFTR), and AQP9 co-localize in the apical membrane of principal cells of the epididymis and the vas deferens, and that both NHERF1 and CFTR co-immunoprecipitate with AQP9. Overlay assays revealed that AQP9 binds to both the PDZ1 and PDZ2 domains of NHERF1, with an apparently higher affinity for PDZ1 versus PDZ2. Pull-down assays showed that the AQP9 COOH-terminal SVIM motif is essential for interaction with NHERF1. Functional assays on isolated tubules perfused in vitro showed a high permeability of the apical membrane to glycerol, which is inhibited by the AQP9 inhibitor, phloretin, and is markedly activated by cAMP. The CFTR inhibitors DPC, GlyH-101 and CFTRinh-172 all significantly reduced the cAMP-activated glycerol-induced cell swelling. We propose that CFTR is an important regulator of AQP9 and that the interaction between AQP9, NHERF1, and CFTR may facilitate the activation of AQP9 by cAMP.
水和溶质跨细胞的质膜运输是一项至关重要的生物学功能，主要由水通道蛋白和水甘油通道蛋白介导。目前对这些膜蛋白的调控仍未完全了解。我们以雄性生殖道作为一个水和甘油运输对生育力建立至关重要的模型系统，现在报告一种调节水通道蛋白9（AQP9）通透性的新途径。AQP9是附睾、肝脏和外周白细胞中的主要水甘油通道蛋白，其COOH末端部分包含一个假定的PDZ结合基序（SVIM）。在这里我们表明，NHERF1、囊性纤维化跨膜传导调节因子（CFTR）和AQP9共定位于附睾主细胞和输精管的顶端膜，并且NHERF1和CFTR都与AQP9进行共免疫沉淀。覆盖分析表明，AQP9与NHERF1的PDZ1和PDZ2结构域都结合，对PDZ1的亲和力明显高于PDZ2。下拉分析表明，AQP9的COOH末端SVIM基序对于与NHERF1的相互作用至关重要。对体外灌注的分离小管进行的功能分析表明，顶端膜对甘油具有高通透性，这被AQP9抑制剂根皮素抑制，并被cAMP显著激活。CFTR抑制剂DPC、GlyH - 101和CFTRinh - 172都显著降低了cAMP激活的甘油诱导的细胞肿胀。我们提出CFTR是AQP9的重要调节因子，并且AQP9、NHERF1和CFTR之间的相互作用可能促进cAMP对AQP9的激活。
