Kasaian M T, Neet K E
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
J Biol Chem. 1988 Apr 15;263(11):5083-90.
Binding and internalization of nerve growth factor (NGF) by responsive cells is a complex process. We have incubated rat pheochromocytoma cells (PC12) with 125I-NGF at 37 degrees C and measured the association of ligand after removal of subsets of bound ligand by different methods. Chase with unlabeled NGF at either 4 or 37 degrees C, acid stripping, nonionic detergent stability, and combinations of these protocols were utilized. These variations of the binding assay were able to distinguish ligand bound to fast versus slow cell surface receptors, NGF bound to slow receptors at the cell surface versus cell interior, and soluble ligand versus cytoskeletally attached NGF. Quantitative and temporal relations among five cellular pools were defined. Experiments with the inhibitors chloroquine, cytochalasin B, and colchicine defined pools of NGF in terms of the route through the cell from the plasma membrane to the lysosome. Chloroquine caused accumulation of NGF only in the pool that was not associated with the cytoskeleton, implicating the involvement of this pool in supplying ligand to the lysosome. Results with cytochalasin B and colchicine suggest that both microfilaments and microtubules are involved in pathways leading to NGF degradation. A semiquantitative model for the movement of NGF through the cell is presented based on these observations.
神经生长因子（NGF）与反应性细胞的结合及内化是一个复杂的过程。我们将大鼠嗜铬细胞瘤细胞（PC12）与125I-NGF在37℃下孵育，并通过不同方法去除结合配体的亚群后测量配体的结合情况。采用在4℃或37℃下用未标记的NGF进行追踪、酸剥离、非离子去污剂稳定性以及这些方案的组合。这些结合测定的变化能够区分与快速和慢速细胞表面受体结合的配体、细胞表面与细胞内部与慢速受体结合的NGF、可溶性配体与细胞骨架附着的NGF。定义了五个细胞池之间的定量和时间关系。用抑制剂氯喹、细胞松弛素B和秋水仙碱进行的实验根据从质膜到溶酶体穿过细胞的途径定义了NGF的池。氯喹仅导致NGF在与细胞骨架不相关的池中积累，这表明该池参与向溶酶体供应配体。细胞松弛素B和秋水仙碱的结果表明，微丝和微管都参与导致NGF降解的途径。基于这些观察结果，提出了一个NGF在细胞内移动的半定量模型。
