Soluble Form of the (Pro) Renin Receptor Generated by Intracellular Cleavage by Furin Is Secreted in Plasma
Soluble Form of the (Pro) Renin Receptor Generated by Intracellular Cleavage by Furin Is Secreted in Plasma
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DOI：
10.1161/hypertensionaha.108.127258
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发表时间：
2009-06-01
期刊：
影响因子：
8.3
通讯作者：
Nguyen, Genevieve
中科院分区：
文献类型：
--
作者：
Cousin, Christelle;Bracquart, Diane;Nguyen, Genevieve
（原）肾素受体[（P）RR]是一种35 kDa的跨膜蛋白，在血管紧张素组织生成和非蛋白水解性原肾素活化中起关键作用。我们在培养细胞的条件培养基中检测到可溶形式的（P）RR [s（P）RR; 28 kDa]。本研究的目的是鉴定负责产生s（P）RR的蛋白酶，即脱落位点，并确定血浆中循环s（P）RR的存在。我们基于以下内容将弗林蛋白酶鉴定为负责内源性（P）RR脱落的蛋白酶：缺乏活性弗林蛋白酶的LoVo结肠癌细胞合成全长（P）RR，但不分泌s（P）RR;用编码α 1-抗胰蛋白酶波特兰变体（一种弗林蛋白酶抑制剂）的质粒转染中国仓鼠卵巢细胞，完全抑制s（P）RR的产生，而在培养基中加入金属蛋白酶抑制剂GM 6001或肿瘤坏死因子-α蛋白酶抑制剂-1（ADAM 17的抑制剂）没有影响;当体外翻译编码（P）RR的cDNA并与重组弗林蛋白酶或ADAM 17孵育时，只有弗林蛋白酶能够产生28 kDa-s（P）RR，在潜在的弗林蛋白酶切割位点R275 A/KT/R278 A上的突变消除了s（P）RR的产生。肾小球上皮细胞的免疫荧光研究表明，（P）RR在trans-Golgi中被切割，与肾素的共沉淀实验表明，s（P）RR存在于血浆中。总之，我们的研究结果表明，s（P）RR产生细胞内的弗林蛋白酶裂解，并在血浆中检测到的s（P）RR能够结合肾素。（高血压。2009;53：1077-1082.）
The (pro) renin receptor [(P) RR] is a 35-kDa transmembrane protein that plays a pivotal role in angiotensin tissue generation and in nonproteolytic prorenin activation. We detected a soluble form of (P) RR [s(P) RR; 28 kDa] in the conditioned medium of cultured cells. The aims of our study were to identify the protease responsible for the generation of s(P) RR, the site of shedding, and to establish the existence of circulating s(P) RR in plasma. We identified furin as the protease responsible for the shedding of endogenous (P) RR based on the following: LoVo colon carcinoma cells devoid of active furin synthesize full-length (P) RR but do not secrete s(P) RR; transfection of Chinese hamster ovary cells with a plasmid coding for alpha 1-antitrypsin Portland variant, an inhibitor of furin, completely inhibited the generation of s( P) RR, whereas addition of GM6001, an inhibitor of metalloproteases or of tumor necrosis factor-alpha protease inhibitor-1, an inhibitor of ADAM17, in the culture medium has no effect; when the cDNA coding for (P) RR was translated in vitro and incubated with recombinant furin or ADAM17, only furin was able to generate the 28 kDa-s(P) RR, and mutagenesis in the potential furin cleavage R275A/KT/R278A site abolished s(P) RR generation. Immunofluorescence study in glomerular epithelial cells showed that (P) RR was cleaved in the trans-Golgi, and coprecipitation experiments with renin showed that s(P) RR was present in plasma. In conclusion, our results show that s(P) RR is generated intracellularly by furin cleavage, and that s(P) RR detected in plasma is able to bind renin. (Hypertension. 2009;53:1077-1082.)