The heteromeric PC-1/PC-2 polycystin complex is activated by the PC-1 N-terminus.
The heteromeric PC-1/PC-2 polycystin complex is activated by the PC-1 N-terminus.
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DOI：
10.7554/elife.60684
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发表时间：
2020-11-09
期刊：
影响因子：
7.7
通讯作者：
Delling M
中科院分区：
文献类型：
--
作者：
Ha K;Nobuhara M;Wang Q;Walker RV;Qian F;Schartner C;Cao E;Delling M
多囊蛋白PC-1和PC-2的突变会导致常染色体显性多囊肾病（ADPKD）并最终导致肾衰竭。PC-1和PC-2富集在初级纤毛上，在那里它们被认为形成异聚离子通道复合物。然而，由于在可靠地测量其活性方面存在技术障碍，对推定的PC-1/PC-2多囊蛋白复合物的功能性理解缺乏。在这里，我们成功地重建的PC-1/PC-2复合物在哺乳动物细胞的质膜，并显示它的功能作为一个外向整流通道。使用重建和纤毛多囊蛋白通道，我们进一步表明，从PC-1的N-末端胞外结构域产生的可溶性片段作为一个内在的激动剂，是必要的和足够的通道激活的功能。因此，我们提出，PC-1的N-末端，ADPKD突变的热点，在体内产生可溶性配体的自体蛋白水解切割。这些发现为理解PC-1/PC-2异聚体在ADPKD中的作用建立了一个机制框架，并提出了新的治疗策略，这些策略将扩展目前可用于这种进行性终末期疾病的有限对症治疗。在大多数动物和其他真核细胞的表面上，都有小的杆状突起，称为初级纤毛。每一个纤毛都被一个特殊的膜包裹着，这个膜富含蛋白质复合物，帮助细胞感知它的局部环境。这些复合物中的一些帮助将离子运输出细胞，而另一些则充当接收化学信号的受体，称为配体。被称为多囊蛋白复合物的独特离子通道能够执行这两种作用，因为它除了包含称为PC-2的离子通道之外还包含称为PC-1的受体。编码PC-1和PC-2的基因中的各种突变可导致常染色体显性多囊肾病（ADPKD），这是人类最常见的单基因疾病。然而，由于初级纤毛的尺寸很小-厚度不到千分之一毫米-对于多囊蛋白复合物如何调节以及突变如何导致ADPKD知之甚少。为了克服这一障碍，Ha等人修改了实验室中生长的肾细胞，使PC-1和PC-2在质膜中形成围绕整个细胞的工作通道。由于细胞的体积大约是纤毛的10，000倍，这使得可以使用常规技术研究离子在多囊蛋白复合物中的运动。使用这种新开发的检测方法进行的实验表明，PC-1蛋白质一端的一个区域（称为C型凝集素结构域）对于刺激多囊蛋白复合物至关重要。Ha等人发现PC-1的这个结构域能够从蛋白质复合物中切割自身。进一步的实验表明，当含有C型凝集素结构域的PC-1片段不再与膜结合时，它们可以激活纤毛和质膜中的多囊蛋白通道。这表明PC-1的这一区域也可能作为一个分泌的配体，可以激活其他多囊蛋白通道。一些导致ADPKD的基因突变可能会破坏多囊蛋白复合物的活性，并降低其跨纤毛膜转运离子的能力。因此，本研究中创建的细胞测定可用于筛选可恢复ADPKD患者中这些离子通道活性的小分子。
Mutations in the polycystin proteins, PC-1 and PC-2, result in autosomal dominant polycystic kidney disease (ADPKD) and ultimately renal failure. PC-1 and PC-2 enrich on primary cilia, where they are thought to form a heteromeric ion channel complex. However, a functional understanding of the putative PC-1/PC-2 polycystin complex is lacking due to technical hurdles in reliably measuring its activity. Here we successfully reconstitute the PC-1/PC-2 complex in the plasma membrane of mammalian cells and show that it functions as an outwardly rectifying channel. Using both reconstituted and ciliary polycystin channels, we further show that a soluble fragment generated from the N-terminal extracellular domain of PC-1 functions as an intrinsic agonist that is necessary and sufficient for channel activation. We thus propose that autoproteolytic cleavage of the N-terminus of PC-1, a hotspot for ADPKD mutations, produces a soluble ligand in vivo. These findings establish a mechanistic framework for understanding the role of PC-1/PC-2 heteromers in ADPKD and suggest new therapeutic strategies that would expand upon the limited symptomatic treatments currently available for this progressive, terminal disease. On the surface of most animal and other eukaryotic cells are small rod-like protrusions known as primary cilia. Each cilium is encased by a specialized membrane which is enriched in protein complexes that help the cell sense its local environment. Some of these complexes help transport ions in out of the cell, while others act as receptors that receive chemical signals called ligands. A unique ion channel known as the polycystin complex is able to perform both of these roles as it contains a receptor called PC-1 in addition to an ion channel called PC-2. Various mutations in the genes that code for PC-1 and PC-2 can result in autosomal dominant polycystic kidney disease (ADPKD), which is the most common monogenetic disease in humans. However, due to the small size of primary cilia – which are less than a thousandth of a millimeter thick – little is known about how polycystin complexes are regulated and how mutations lead to ADPKD. To overcome this barrier, Ha et al. modified kidney cells grown in the lab so that PC-1 and PC-2 form a working channel in the plasma membrane which surrounds the entire cell. As the body of a cell is around 10,000 times bigger than the cilium, this allowed the movement of ions across the polycystin complex to be studied using conventional techniques. Experiments using this newly developed assay revealed that a region at one of the ends of the PC-1 protein, named the C-type lectin domain, is essential for stimulating polycystin complexes. Ha et al. found that this domain of PC-1 is able to cut itself from the protein complex. Further experiments showed that when fragments of PC-1, which contain the C-type lectin domain, are no longer bound to the membrane, they can activate the polycystin channels in cilia as well as the plasma membrane. This suggests that this region of PC-1 may also act as a secreted ligand that can activate other polycystin channels. Some of the genetic mutations that cause ADPKD likely disrupt the activity of the polycystin complex and reduce its ability to transport ions across the cilia membrane. Therefore, the cell assay created in this study could be used to screen for small molecules that can restore the activity of these ion channels in patients with ADPKD.