In situ detection of phosphorylated platelet-derived growth factor receptor β using a generalized proximity ligation method
In situ detection of phosphorylated platelet-derived growth factor receptor β using a generalized proximity ligation method
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DOI：
10.1074/mcp.m700166-mcp200
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发表时间：
2007-09-01
影响因子：
7
通讯作者：
Soderberg, Ola
中科院分区：
文献类型：
--
作者：
Jarvius, Malin;Paulsson, Janna;Soderberg, Ola
需要改进的方法来原位表征细胞系和组织中的翻译后修饰。例如，期望监测来自用抑制剂治疗的人肿瘤的样品中的个体受体酪氨酸激酶的磷酸化状态以评价治疗反应。不幸的是，用于原位观察组织翻译后修饰的动力学的主要方法，免疫组织化学和免疫荧光，表现出有限的灵敏度和选择性。邻位连接分析是一种新的方法，其通过双重识别的要求提供改进的选择性，并且通过包括DNA扩增作为靶分子检测的组分来增加灵敏度。因此，我们建立了一种通用的原位邻位连接试验，以研究血小板衍生生长因子BB刺激的细胞中血小板衍生生长因子受体β（PDGFR β）的磷酸化.将通过连接DNA链修饰的对来自不同物种的免疫球蛋白具有特异性的抗体与来自相应物种的一对一抗一起用作二次邻近探针。受体和磷酸化位点的双重识别通过与第二邻近探针组合的第一抗体用于产生环状DNA链;这之后通过经由滚环扩增复制DNA环进行信号扩增。我们在用β-受体转染的猪主动脉内皮细胞中稳定过表达人流感血凝素标记的人PDGFR β的人胚肾细胞中检测到酪氨酸磷酸化的PDGFR β，但在用α-受体转染的细胞中没有，并且在内源性表达PDGFR β的永生化人包皮成纤维细胞BJhTert中也检测到酪氨酸磷酸化的PDGFR β.我们还观察了来自新鲜冷冻的经历伤口愈合的人瘢痕组织的组织切片中酪氨酸磷酸化的PDGFR β。该方法对于研究信号传导、筛选药物作用以及增强组织病理学诊断潜力具有重要价值。
Improved methods are needed for in situ characterization of post- translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post- translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet- derived growth factor receptor beta ( PDGFR beta) in cells stimulated with platelet- derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFR beta in human embryonic kidney cells stably overexpressing human influenza hemagglutinin- tagged human PDGFR beta in porcine aortic endothelial cells transfected with the beta- receptor, but not in cells transfected with the alpha- receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFR beta.. We furthermore visualized tyrosine phosphorylated PDGFR beta in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.