RasGRP1 stimulation enhances ubiquitination and endocytosis of the sodium-chloride cotransporter
RasGRP1 stimulation enhances ubiquitination and endocytosis of the sodium-chloride cotransporter
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DOI：
10.1152/ajprenal.00441.2009
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发表时间：
2010-08-01
影响因子：
4.2
通讯作者：
Hoover, Robert S.
中科院分区：
文献类型：
--
作者：
Ko, Benjamin;Kamsteeg, Erik-Jan;Hoover, Robert S.
李建军，李建军，李建军，李建军，等。rgrp1基因的表达及其调控作用。[J] .中国生物医学工程学报，2016,31(3):557 - 557。首次发表于2010年4月14日；doi: 10.1152 / ajprenal.00441.2009。氯化钠共转运体（NCC）是远曲小管中主要的盐吸收途径。最近，我们描述了一种新的NCC调控途径，即phorbol酯（PE）刺激Ras guanyl-releasing protein 1 (RasGRP1)，触发级联最终激活ERK1/2 MAPK并降低NCC细胞表面表达（Ko B, Joshi m, Cooke LL, Vazquez N, Musch MW, Hebert SC, Gamba G, Hoover RS. pronatalacrsci, 2007）。对这些影响NCC活性的机制知之甚少。通过改变NCC表面表达来调节NCC已经有报道，但NCC的内吞作用尚未得到证实。在这项研究中，我们利用生物素化、内化分析和动力蛋白显性-负结构，证明PE对NCC的调节是通过NCC内化的增强而发生的，并且依赖于动力蛋白。此外，NCC的免疫沉淀和随后的泛素免疫印迹显示，phopbol酯处理增加了NCC的泛素化。MEK1/2抑制剂和RasGRP1的基因沉默表明这种作用依赖于RasGRP1和ERK1/2的激活。抑制泛素化可防止pe介导的NCC表面表达下降（通过生物素化测量）或NCC活性（通过放射性示踪剂摄取测量）。这些发现证实了PE对NCC的作用是通过NCC的内吞作用介导的。此外，NCC的泛素化对这一过程至关重要，而这种泛素化依赖于rasgrp1介导的ERK1/2激活。
Ko B, Kamsteeg E, Cooke LL, Moddes LN, Deen PM, Hoover RS. RasGRP1 stimulation enhances ubiquitination and endocytosis of the sodium-chloride cotransporter. Am J Physiol Renal Physiol 299: F300-F309, 2010. First published April 14, 2010; doi:10.1152/ajprenal.00441.2009.-The sodium-chloride cotransporter (NCC) is the principal salt-absorptive pathway in the distal convoluted tubule. Recently, we described a novel pathway of NCC regulation in which phorbol esters (PE) stimulate Ras guanyl-releasing protein 1 (RasGRP1), triggering a cascade ultimately activating ERK1/2 MAPK and decreasing NCC cell surface expression (Ko B, Joshi LM, Cooke LL, Vazquez N, Musch MW, Hebert SC, Gamba G, Hoover RS. Proc Natl Acad Sci USA 104: 20120-20125, 2007). Little is known about the mechanisms which underlie these effects on NCC activity. Regulation of NCC via changes in NCC surface expression has been reported, but endocytosis of NCC has not been demonstrated. In this study, utilizing biotinylation, internalization assays, and a dynamin dominant-negative construct, we demonstrate that the regulation of NCC by PE occurs via an enhancement in internalization of NCC and is dynamin dependent. In addition, immunoprecipitation of NCC and subsequent immunoblotting for ubiquitin showed increased ubiquitination of NCC with phorbol ester treatment. MEK1/2 inhibitors and gene silencing of RasGRP1 indicated that this effect was dependent on RasGRP1 and ERK1/2 activation. Inhibition of ubiquitination prevents any PE-mediated decrease in NCC surface expression as measured by biotinylation or NCC activity as measured by radiotracer uptake. These findings confirmed that the PE effect on NCC is mediated by endocytosis of NCC. Furthermore, ubiquitination of NCC is essential for this process and this ubiquitination is dependent upon RasGRP1-mediated ERK1/2 activation.