Department of Endocrinology, Diabetes and Metabolism, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa, 252-0374, Japan.
Protosera Inc., 4-3-22 Nishinakajima, Yodogawa-ku, Osaka, 532-0011, Japan.
Sci Rep. 2018 Dec 14;8(1):17865. doi: 10.1038/s41598-018-35740-6.
Although orphan G protein-coupled receptors (GPCRs) have been used as targets to discover unidentified natural ligands, increasing numbers of non-GPCRs have been found to mediate important biological functions. Bioinformatics of genome and cDNA resources predict putative bioactive peptides, demanding an alternative approach to efficiently unravel cell surface targets. In silico analysis of a full-length cDNA library previously allowed us to identify salusin-β, a parasympathomimetic/pro-atherosclerotic peptide with unique physicochemical properties. Here, we show that the β-chain of ATP synthase is a cell surface receptor for salusin-β by utilizing artificial liposomes embedded with endogenous membrane proteins directly transferred from animal tissues while retaining the ligand-binding capability. Conventional techniques using detergents identified a β-actin-profilin complex as membrane-associated salusin-β-binding proteins, but failed to identify the cell surface receptor. Since the α-chain of ATP synthase is a principal cell surface target for angiostatin, a potent endogenous angiogenesis inhibitor, we investigated whether salusin-β modulates angiogenesis. Salusin-β inhibited cell surface ATP synthase activity and prevented sarcoma cell-induced angiogenesis in an in vivo mouse air sac model. Therefore, salusin-β binds to membrane-bound ATP synthase and acts as an angiogenesis inhibitor. The current methodology allows the identification of novel cell surface targets, irrespective of the receptor structure.
虽然孤儿 G 蛋白偶联受体（GPCRs）已被用作发现未知天然配体的靶点，但越来越多的非 GPCR 被发现介导重要的生物学功能。基因组和 cDNA 资源的生物信息学预测了潜在的生物活性肽，这需要一种替代方法来有效地揭示细胞表面靶点。我们之前通过全长 cDNA 文库的计算分析，鉴定出了 salusin-β，这是一种具有独特理化性质的拟副交感/动脉粥样硬化肽。在这里，我们通过利用直接从动物组织中转录的内源性膜蛋白嵌入人工脂质体，同时保留配体结合能力，证明 ATP 合酶的β 链是 salusin-β 的细胞表面受体。使用去污剂的传统技术鉴定了β-肌动蛋白-丝氨酸羟甲基转移酶复合物作为与膜相关的 salusin-β 结合蛋白，但未能鉴定出细胞表面受体。由于 ATP 合酶的α 链是血管抑素（一种有效的内源性血管生成抑制剂）的主要细胞表面靶标，我们研究了 salusin-β 是否调节血管生成。Salusin-β 抑制细胞表面 ATP 合酶活性，并防止肉瘤细胞在体内气囊模型中诱导血管生成。因此，salusin-β 与膜结合的 ATP 合酶结合，并作为一种血管生成抑制剂发挥作用。当前的方法学允许鉴定新的细胞表面靶点，而与受体结构无关。
