Subramanian Shyamsundar, Tsai Richard, Sen Shamik, Dahl Kris Noel, Discher Dennis E
Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
Blood Cells Mol Dis. 2006 May-Jun;36(3):364-72. doi: 10.1016/j.bcmd.2006.01.012. Epub 2006 May 11.
Integrin Associated Protein (IAP, CD47) is a ubiquitous integral membrane protein implicated in processes (in mice) that range from inhibiting clearance by phagocytes [Oldenborg et al., Science 2000; Gardai et al., Cell 2005] to neutrophil motility [Lindberg et al., Science 1996]. SIRPalpha is CD47's main receptor on phagocytes plus a number of other cell types, and SIRPalpha-CD47 interactions in clusters are believed to mediate signaling. However, considerable species differences in CD47 sequence as well as differences in CD47 extractability from mouse cells versus man motivate a characterization of mobility, clusterability, and kinetics under force of CD47-SIRPalpha. Despite similar levels of CD47 on red cells from mouse and man, we find an effective avidity of SIRPalpha-CD47 for mouse appears higher than for human. Both mouse and human CD47 show clustering by multivalent SIRPalpha complexes, but only mouse cells aggregate with CD47 concentrating at cell-cell contacts. This proves consistent with fluorescence imaged micro-deformation, which indicates near-complete mobility of CD47 on mouse cells compared to only about 30-40% mobility on normal human cells. To qualify the method, we also show that disrupting cellular F-actin dramatically increases the mobility of integral membrane proteins. Furthermore, atomic force microscopy probing of cell membranes with human SIRPalpha confirms the species-specific interactions and provides evidence of clustering and adhesion on short time scales, but it also shows surprisingly strong forces in detachment for a signaling complex. The results thus highlight major species differences in CD47-SIRPalpha interactions and CD47 integration, suggesting that signaling by CD47 in man may be qualitatively different from mouse.
整合素相关蛋白（IAP，CD47）是一种普遍存在的整合膜蛋白，在（小鼠的）多种过程中发挥作用，这些过程包括抑制吞噬细胞的清除作用[奥尔登伯格等人，《科学》，2000年；加尔代等人，《细胞》，2005年]以及中性粒细胞的运动[林德伯格等人，《科学》，1996年]。信号调节蛋白α（SIRPα）是吞噬细胞以及许多其他细胞类型上CD47的主要受体，并且成簇的SIRPα - CD47相互作用被认为可介导信号传导。然而，CD47序列存在相当大的物种差异，以及从小鼠细胞与人细胞中提取CD47的差异，这促使人们对CD47 - SIRPα在力作用下的流动性、成簇能力和动力学进行表征。尽管小鼠和人的红细胞上CD47水平相似，但我们发现SIRPα - CD47对小鼠的有效亲和力似乎高于对人的。小鼠和人CD47都显示出被多价SIRPα复合物聚集，但只有小鼠细胞聚集，CD47集中在细胞 - 细胞接触部位。这与荧光成像微变形结果一致，该结果表明CD47在小鼠细胞上几乎完全可移动，而在正常人细胞上只有约30 - 40%的可移动性。为验证该方法，我们还表明破坏细胞的F - 肌动蛋白会显著增加整合膜蛋白的流动性。此外，用人SIRPα对细胞膜进行原子力显微镜探测证实了物种特异性相互作用，并提供了短时间尺度上成簇和粘附的证据，但它也显示出对于一个信号复合物而言，分离时的力惊人地强。因此，这些结果突出了CD47 - SIRPα相互作用和CD47整合中的主要物种差异，表明人CD47的信号传导在性质上可能与小鼠不同。
