Buxser S E, Watson L, Johnson G L
J Cell Biochem. 1983;22(4):219-33. doi: 10.1002/jcb.240220404.
Rat PC12 pheochromocytoma and human A875 melanoma cells express nerve growth factor (NGF) receptors on their surfaces. Covalent crosslinking of bound 125I-NGF to PC12 or A875 intact cells or plasma membrane-enriched fractions resulted in labelling of a peptide doublet at Mr = 110,000 and a single labelled peptide at Mr = 200,000 for each of the cell and membrane preparations. However, a difference between equilibrium binding properties of NGF-receptor on PC12 and A875 cells was observed. PC12 cells exhibited biphasic binding properties with two apparent binding sites: KD = 5.2 nM sites and KD = 0.3 nM sites. The high-affinity PC12 binding sites were trypsin resistant, and 125I-NGF dissociated slowly from them. A875 cells exhibited sites with homogeneous properties (KD = 1.0 nM), all binding sites were trypsin sensitive, and 125I-NGF dissociated rapidly in the presence of unlabelled NGF. Membrane-enriched fractions from either cell type contained binding sites with a uniform low affinity (KD = 3 nM) that were trypsin sensitive, and 125I-NGF rapidly dissociated from them. Sixty to 80 percent of binding sites in membranes could be converted to the high-affinity, trypsin-resistant state by addition of wheat germ agglutinin (WGA). The loss of high-affinity, trypsin-resistant sites from PC12 cells during preparation of plasma membrane fractions does not appear to be the result of selective isolation of low-affinity sites or proteolytic degradation since there is a loss of 125I-NGF binding immediately after cell lysis which is not blocked by protease inhibitors. Also, high-affinity, trypsin-resistant binding sites are not found associated with other cell fractions. The differences between receptor properties on PC12 cells and on A875 cells apparently are the result of differences in the respective intracellular environments. Thus, significant structural homology exists between receptors on A875 and PC12 cells. Cell components other than the binding unit of the NGF receptor may be responsible for the different properties of receptor.
大鼠嗜铬细胞瘤PC12细胞和人黑色素瘤A875细胞在其表面表达神经生长因子（NGF）受体。将结合的¹²⁵I-NGF与PC12或A875完整细胞或富含质膜的组分进行共价交联，结果显示，对于每种细胞和膜制剂，在相对分子质量（Mr）= 110,000处标记出一个双肽，在Mr = 200,000处标记出一个单肽。然而，观察到PC12细胞和A875细胞上NGF受体的平衡结合特性存在差异。PC12细胞表现出双相结合特性，有两个明显的结合位点：解离常数（KD）= 5.2 nM的位点和KD = 0.3 nM的位点。PC12细胞的高亲和力结合位点对胰蛋白酶具有抗性，¹²⁵I-NGF从这些位点缓慢解离。A875细胞表现出具有均一特性的位点（KD = 1.0 nM），所有结合位点对胰蛋白酶敏感，并且在未标记的NGF存在下，¹²⁵I-NGF迅速解离。来自任何一种细胞类型的富含膜的组分都含有具有均匀低亲和力（KD = 3 nM）的结合位点，这些位点对胰蛋白酶敏感，¹²⁵I-NGF从它们上面迅速解离。通过添加麦胚凝集素（WGA），膜中60%至80%的结合位点可转变为高亲和力、对胰蛋白酶具有抗性的状态。在制备质膜组分过程中，PC12细胞上高亲和力、对胰蛋白酶具有抗性的位点的丧失似乎不是选择性分离低亲和力位点或蛋白水解降解的结果，因为细胞裂解后立即出现¹²⁵I-NGF结合的丧失，而这并未被蛋白酶抑制剂所阻断。此外，未发现高亲和力、对胰蛋白酶具有抗性的结合位点与其他细胞组分相关联。PC12细胞和A875细胞上受体特性的差异显然是各自细胞内环境差异的结果。因此，A875细胞和PC12细胞上的受体之间存在显著的结构同源性。NGF受体结合单元以外的细胞成分可能是受体不同特性的原因。
