Isolation and characterization of detergent-resistant microdomains responsive to NCAM-mediated signaling from growth cones
Isolation and characterization of detergent-resistant microdomains responsive to NCAM-mediated signaling from growth cones
复制标题
DOI：
10.1006/mcne.2001.1060
复制
发表时间：
2002-01-01
影响因子：
3.5
通讯作者：
Meiri, KF
中科院分区：
文献类型：
--
作者：
He, Q;Meiri, KF
目前尚不清楚细胞粘附分子（CAM）介导的信号传导如何引起生长锥细胞骨架的反应。在这里，我们使用TX-114提取的生长锥，然后通过平衡梯度离心分离的洗涤剂抗性微区（DRM），可以在结构和功能上区分的基础上的本地化和激活CAM介导的信号通路的组件的亚组分。富含胆固醇、小窝蛋白、NCAM 140、GPI连接的NCAM 120、fyn和GAP-43的DRM（所有微结构域或筏的常规标记物）位于梯度的区域2和3中。通过GAP-43对CAM信号通路的特定组分进行免疫共沉淀，然后鉴定了DRM的不同亚群。来自区域2 DRM的GAP-43共沉淀GPI连接的NCAM 120并且是无活性的，即，PKC磷酸化没有被刺激。相反，来自区域3 DRM的GAP-43共沉淀跨膜NCAM 140和小窝蛋白，并且是活性的，即，被PKC高度磷酸化。来自区域2和区域3的DRM的不同子集含有不能与GAP-43抗体共沉淀的fyn。在这种情况下，区域2 DRM含有在Y 415上磷酸化的活化fyn。相反，区域3 DRM包含不活跃的fyn。因此，fyn和GAP-43这两个NCAM信号传导的靶标位于不同的DRM群体中，并且它们的活化形式在梯度上均匀分布。从4号区域回收的抗洗涤剂膜部分富含NCAM 140、磷酸化GAP-43和肌动蛋白，但不富含胆固醇、小窝蛋白或fyn。免疫电镜显示磷酸化GAP-43定位于膜和F-肌动蛋白相互作用的地方。我们的研究结果为NCAM介导的DRMs信号转导提供了证据，并表明负责fyn和PKC/GAP-43介导的NCAM信号转导的DRMs在结构上是不同的，并且差异性地分布在生长锥中。
It is still largely unclear how cell adhesion molecule (CAM)-mediated signaling evokes responses from the growth cone cytoskeleton. Here we used TX-114 extraction of growth cones followed by equilibrium gradient centrifugation to isolate subfractions of detergent-resistant microdomains (DRMs) that could be structurally and functionally distinguished on the basis of localization and activation of components of CAM-mediated signaling pathways. DRMs enriched in cholesterol, caveolin, NCAM140, GPI-linked NCAM120, fyn, and GAP-43, all conventional markers of microdomains or rafts, were located in areas 2 and 3 of the gradient. Coimmunoprecipitation of specific components of CAM signaling pathways by GAP-43 then identified distinct subpopulations of DRMs. GAP-43 from area 2 DRMs coprecipitated GPI-linked NCAM120 and was inactive, i.e., PKC phosphorylation had not been stimulated. In contrast the GAP-43 from area 3 DRMs coprecipitated both transmembrane NCAM140 and caveolin and was active, i.e., highly phosphorylated by PKC. A different subset of DRMs from both area 2 and area 3 contained fyn that could not be coprecipitated with GAP-43 antibodies. In this case area 2 DRMs contained activated fyn that was phosphorylated on Y415. In contrast area 3 DRMs contained inactive fyn. Hence fyn and GAP-43, both targets of NCAM signaling, are located in distinct populations of DRMs, and their activated forms are reciprocally distributed on the gradient. A detergent-resistant membrane fraction recovered from area 4 was enriched in NCAM140, phosphorylated GAP-43, and actin, but not cholesterol, caveolin, or fyn. Immunoelectron microscopy revealed that phosphorylated GAP-43 was localized where the membranes and F-actin interacted. Our results provide evidence for NCAM-mediated signaling in DRMs and suggest that the DRMs responsible for fyn and PKC/GAP-43-mediated NCAM signaling are structurally distinct and differentially distributed in growth cones.