NERVE GROWTH-FACTOR RECEPTORS ON PC12 CELLS - EVIDENCE FOR 2 RECEPTOR CLASSES WITH DIFFERING CYTOSKELETAL ASSOCIATION
NERVE GROWTH-FACTOR RECEPTORS ON PC12 CELLS - EVIDENCE FOR 2 RECEPTOR CLASSES WITH DIFFERING CYTOSKELETAL ASSOCIATION
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DOI：
10.1016/0092-8674(81)90112-4
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发表时间：
1981-01-01
期刊：
影响因子：
64.5
通讯作者：
BOTHWELL, MA
中科院分区：
文献类型：
--
作者：
SCHECHTER, AL;BOTHWELL, MA
大鼠嗜铬细胞瘤PC 12细胞是一种NGF [神经生长因子]反应细胞系，具有2类NGF受体，分别称为快受体和慢受体。占特异性NGF结合的75%的快受体的特征在于其快速的125 I-NGF结合和解离速率。在37度。C，125 I-NGF与快受体的结合比与慢受体的结合快5倍，125 I-NGF从快受体的解离比从慢受体的解离快40倍。没有证据表明配体诱导的受体从快到慢的特征转换。结合实验的Scatchard分析表明，PC 12细胞具有60，000个NGF特异性受体，其中15，000个是慢型受体。尽管具有非常不同的动力学常数，但慢和快受体具有相似的平衡结合常数（约2倍。10-10 M），这是由于不同缔合和解离速率的抵消效应。在添加NGF之前用胰蛋白酶短暂消化PC 12细胞基本上使所有快受体失活，而不显著影响慢受体。因此，在加入NGF之前必须存在快和慢受体类别，并且观察到的受体异质性不是由于配体诱导的变化。与慢受体结合的125 I-NGF优先与Triton X-100不溶性细胞骨架的制备物相关，而与快受体结合的125 I-NGF通过该方法溶解。细胞内相关的神经生长因子几乎只与细胞核内的细胞骨架基质相关，而不与细胞核本身相关。通过各种方法制备的细胞核表明，在评估是否存在移位和NGF与细胞核的结合时，应考虑污染细胞骨架元素的存在。通过将温度降低到0 ℃或更低来抑制NGF的内吞内化。C或在缺乏葡萄糖的培养基中用叠氮化钠预孵育细胞不会减少结合的NGF的缓慢释放组分或改变其细胞骨架结合。慢和细胞骨架受体可能的功能作用进行了讨论。
Rat pheochromocytoma PC12 cells, an NGF [nerve growth factor] responsive cell line, exhibit 2 classes of NGF receptors which were designated Fast and Slow. Fast receptors, accounting for 75% of specific NGF binding, are distinguished by their rapid rates for association and dissociation of 125I-NGF. At 37.degree. C, binding of 125I-NGF to Fast receptors is 5-fold more rapid than to Slow receptors and dissociation of 125I-NGF from Fast receptors is 40-fold more rapid than from Slow receptors. No evidence was obtained for a ligand-induced conversion of receptors from Fast to Slow characteristics. Scatchard analysis of binding experiments indicates that PC12 cells possess 60,000 specific receptors for NGF of which 15,000 are of the Slow class. Despite having very different kinetic constants, Slow and Fast receptors have similar equilibrium binding constants (.apprx. 2 .times. 10-10 M) due to cancelling effects of differing association and dissociation rates. Brief digestion of PC12 cells with trypsin before addition of NGF inactivates essentially all Fast receptors without significantly affecting Slow receptors. Therefore Fast and Slow classes of receptors must exist prior to addition of NGF, and the observed receptor heterogeneity is not due to ligand-induced changes. 125I-NGF bound to Slow receptors is preferentially associated with preparations of Triton X-100 insoluble cytoskeletons, while 125I-NGF bound to Fast receptors is solubilized by this procedure. Cytoskeletally associated NGF is almost exclusively associated with the extranuclear cytoskeletal matrix rather than with the nucleus itself. Preparation of nuclei by various methods suggests that the presence of contaminating cytoskeletal elements should be considered in evaluating the existence of translocation and binding of NGF to the nucleus. Inhibition of endocytotic internalization of NGF either by lowering of temperature to 0.degree. C or by preincubation of cells with sodium azide in medium lacking glucose does not reduce the slowly released component of bound NGF or alter its cytoskeletal association. The possible functional roles of Slow and cytoskeletal receptors are discussed.