Yankner B A, Shooter E M
Proc Natl Acad Sci U S A. 1979 Mar;76(3):1269-73. doi: 10.1073/pnas.76.3.1269.
Cells of a continuous line of rat pheochromocytoma (PC12) were incubated with 125I-labeled beta nerve growth factor (beta NGF), and at given time intervals the cell nuclei were isolated by a procedure that used the detergent Triton X-100. NGF was detectable in the nucleus after 20 min and continued to accumulate in a linear fashion for several hours after the total binding to the cell had reached steady state. After 17 hr at 37 degrees C, about 60% of the NGF bound to the cell was in the nucleus, NGF was not translocated to the nucleus at 4 degrees C. When nuclei were purified from PC12 cells and incubated with 125I-labeled beta NGF, specific binding sites were found. Binding was saturable and consistent with two sites: a high-affinity site with a Kd of 0.08 nM (+/- nM) and a lower-affinity site with a Kd of 9.0 nM (+/- 2.0 nM). The receptors in the nucleus were shown to be localized to the nuclear membrane. Membrane-free chromatin did not bind NGF specifically. The translocation of NGF to the nucleus was accompanied by a commensurate decrease in the cell-surface binding capacity. In the nucleus, however, the receptor capacities of both sites were increased when PC12 cells were grown in the presence of NGF.
将大鼠嗜铬细胞瘤（PC12）连续细胞系的细胞与125I标记的β神经生长因子（βNGF）一起孵育，在给定的时间间隔，通过使用去污剂曲拉通X-100的方法分离细胞核。20分钟后在细胞核中可检测到NGF，并且在与细胞的总结合达到稳态后，NGF继续以线性方式积累数小时。在37℃孵育17小时后，结合到细胞上的NGF约60%在细胞核中，在4℃时NGF不会转运到细胞核中。当从PC12细胞中纯化细胞核并与125I标记的βNGF一起孵育时，发现了特异性结合位点。结合是可饱和的，符合两个位点：一个高亲和力位点，Kd为0.08 nM（±nM），一个低亲和力位点，Kd为9.0 nM（±2.0 nM）。细胞核中的受体显示定位于核膜。无膜染色质不特异性结合NGF。NGF向细胞核的转运伴随着细胞表面结合能力的相应降低。然而，在细胞核中，当PC12细胞在NGF存在下生长时，两个位点的受体能力均增加。
