目标:贩运,膜保留,Na+/H+交换体3(NHE3)的信号特异性调节由PDZ-接头蛋白的Na+/H+交换体调节因子(NHERF)家族调节。这项研究探索了NHE3和NHERF2与耐去污剂膜微结构域(DRMs,“脂筏”)在小鼠小肠中的体内鸟苷酸循环C受体(Gucy2c)激活过程中。
方法:从野生型中分离小肠刷状缘膜(siBBMs),NHE3缺陷,cGMP激酶II缺陷型,和NHERF2缺陷小鼠,口服耐热大肠杆菌毒素(STa)类似物利那洛肽后。通过Optiprep密度梯度离心法分离TritonX溶解的siBBMs的脂筏和非筏部分。进行共聚焦显微镜检查以研究体内应用利那洛肽后的NHE3再分布。
结果:在WTsiBBM中,NHE3,NHERF2和cGKII与移植物密切相关。NHE3的筏关联,而不是cGKII,是NHERF2依赖的。在将利那洛肽应用于WT小鼠后,NHE3的脂筏结合减少,cGKII的增加,而NHERF2没有改变。BBM中的NHE3表达从微绒毛转移到末端网区域。在cGKII缺陷小鼠中,利那洛肽诱导的NHE3移植物结合和微绒毛丰度的降低被废除,并在NHERF2缺陷小鼠中强烈减少。
结论:NHE3,cGKII,和NHERF2在siBBM中形成脂筏相关信号复合物,它通过Gucy2c活化介导抑制盐和水的吸收。NHERF2增强NHE3的移植物缔合,这对于其与专有移植物相关的活化cGKII的紧密相互作用至关重要。
Trafficking, membrane retention, and signal-specific regulation of the Na+/H+ exchanger 3 (NHE3) are modulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adapter proteins. This study explored the assembly of NHE3 and NHERF2 with the cGMP-dependent kinase II (cGKII) within detergent-resistant membrane microdomains (DRMs, \"lipid rafts\") during in vivo guanylate cycle C receptor (Gucy2c) activation in murine small intestine.
Small intestinal brush border membranes (siBBMs) were isolated from wild type, NHE3-deficient, cGMP-kinase II-deficient, and NHERF2-deficient mice, after oral application of the heat-stable Escherichia coli toxin (STa) analog linaclotide. Lipid raft and non-raft fractions were separated by Optiprep density gradient centrifugation of Triton X-solubilized siBBMs. Confocal microscopy was performed to study NHE3 redistribution after linaclotide application in vivo.
In the WT siBBM, NHE3, NHERF2, and cGKII were strongly raft associated. The raft association of NHE3, but not of cGKII, was NHERF2 dependent. After linaclotide application to WT mice, lipid raft association of NHE3 decreased, that of cGKII increased, while that of NHERF2 did not change. NHE3 expression in the BBM shifted from a microvillar to a terminal web region. The linaclotide-induced decrease in NHE3 raft association and in microvillar abundance was abolished in cGKII-deficient mice, and strongly reduced in NHERF2-deficient mice.
NHE3, cGKII, and NHERF2 form a lipid raft-associated signal complex in the siBBM, which mediates the inhibition of salt and water absorption by Gucy2c activation. NHERF2 enhances the raft association of NHE3, which is essential for its close interaction with the exclusively raft-associated activated cGKII.