School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan, NSW 2308, Australia; Hunter Medical Research Institute, New Lambton Heights, NSW 2305, Australia.
Department of Internal Medicine, Knappschaftskrankenhaus, Ruhr-University Bochum, Bochum, Germany.
Exp Cell Res. 2014 Apr 15;323(1):41-55. doi: 10.1016/j.yexcr.2014.02.012. Epub 2014 Feb 18.
Fat1 is a single pass transmembrane protein and the largest member of the cadherin superfamily. Mouse knockout models and in vitro studies have suggested that Fat1 influences cell polarity and motility. Fat1 is also an upstream regulator of the Hippo pathway, at least in lower vertebrates, and hence may play a role in growth control. In previous work we have established that FAT1 cadherin is initially cleaved by proprotein convertases to form a noncovalently linked heterodimer prior to expression on the cell surface. Such processing was not a requirement for cell surface expression, since melanoma cells expressed both unprocessed FAT1 and the heterodimer on the cell surface. Here we further establish that the site 1 (S1) cleavage step to promote FAT1 heterodimerisation is catalysed by furin and we identify the cleavage site utilised. For a number of other transmembrane receptors that undergo heterodimerisation the S1 processing step is thought to occur constitutively but the functional significance of heterodimerisation has been controversial. It has also been generally unclear as to the significance of receptor heterodimerisation with respect to subsequent post-translational proteolysis that often occurs in transmembrane proteins. Exploiting the partial deficiency of FAT1 processing in melanoma cells together with furin-deficient LoVo cells, we manipulated furin expression to demonstrate that only the heterodimer form of FAT1 is subject to cleavage and subsequent release of the extracellular domain. This work establishes S1-processing as a clear functional prerequisite for ectodomain shedding of FAT1 with general implications for the shedding of other transmembrane receptors.
Fat1 是一种单次跨膜蛋白，也是钙粘蛋白超家族中最大的成员。鼠敲除模型和体外研究表明，Fat1 影响细胞极性和运动。Fat1 也是 Hippo 通路的上游调节剂，至少在低等脊椎动物中如此，因此可能在生长控制中发挥作用。在之前的工作中，我们已经确定 FAT1 钙粘蛋白最初被蛋白水解酶切割，形成非共价连接的异二聚体，然后再表达在细胞表面。这种加工不是细胞表面表达的要求，因为黑色素瘤细胞在细胞表面表达未加工的 FAT1 和异二聚体。在这里，我们进一步确定 1 号位点（S1）切割步骤促进 FAT1 异二聚化是由 furin 催化的，并且我们确定了所使用的切割位点。对于其他一些经历异二聚化的跨膜受体，S1 加工步骤被认为是组成性的，但异二聚化的功能意义一直存在争议。对于跨膜蛋白中经常发生的随后的翻译后蛋白水解，受体异二聚化的意义也通常不清楚。利用黑色素瘤细胞中 FAT1 加工的部分缺陷以及缺乏 furin 的 LoVo 细胞，我们操纵 furin 表达，证明只有 FAT1 的异二聚体形式才会被切割，并随后释放细胞外结构域。这项工作确立了 S1 加工作为 FAT1 胞外结构域脱落的明确功能前提，对其他跨膜受体的脱落具有普遍意义。
