Kinetics of internalization and recycling of the asialoglycoprotein receptor in a hepatoma cell line.
Kinetics of internalization and recycling of the asialoglycoprotein receptor in a hepatoma cell line.
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DOI：
10.1016/s0021-9258(18)34710-0
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发表时间：
1982-04
期刊：
影响因子：
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通讯作者：
A. Schwartz;S. E. Fridovich;H. Lodish
中科院分区：
文献类型：
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作者：
A. Schwartz;S. E. Fridovich;H. Lodish
在人肝癌细胞系Hep 6 - 2中存在大量的功能性去唾液酸糖蛋白受体，这使我们能够确定定义配体与表面受体结合，配体受体内化和受体回收的参数。我们已经开发了一个简单的动力学模型，其中包括所有这些过程。这些细胞含有226，000个功能性脱唾液酸糖蛋白受体，并且在37 ℃下，在半饱和浓度（2 μ g/ml）的251-脱唾液酸类粘蛋白（ASOR）的摄取以15，在4 ℃下261-ASOR结合的速率和在37 ℃下261-ASOR摄取的速率均不大于1000分子/细胞/分钟。C在蛋白质合成被完全抑制的条件下被改变。因此，每种受体平均每16分钟与配体结合一次。在10-12 m-叠氮化物存在下，测量37 ℃下1251-ASOR与细胞的结合速率，与内化无关。在37 ℃下的内化速率是从在4 ℃下与配体预结合的细胞中通过遵循EDTA抗性细胞相关的”% ASOR“来测定的。在用胰蛋白酶预处理以除去所有表面受体的细胞中，从4 ℃下的配体结合和37 ℃下的配体摄取评估细胞内功能性受体的分布。在生长的Hep G2细胞中，87%的受体位于细胞表面，其中超过88%未被配体占据。在1-ASOR的稳态内化期间，表面ASOR受体的数量减少17%。在2 μ g/ml的‘261-ASOR下，未被占据的表面受体需要8.7分钟的平均时间来结合配体，并且需要2.2-2.3分钟的平均时间来内化表面受体-配体复合物。另外需要4.2分钟配体从内化的受体解离并使受体返回细胞表面。我们已经能够通过两组或多组独立的测量来确定或计算受体激动剂的大多数参数。
The presence of a large number of functional asialoglycoprotein receptors in the human hepatoma cell line Hep 6 2 has allowed us to determine the parameters which define ligand binding to the surface receptor, ligand receptor internalization, and receptor recycling. We have developed a simple kinetic model which encompasses all of these processes. These cells contain 226,000 functional asialoglycoprotein receptors, and at 37" C uptake at a half-saturating concentration (2 pg/ml) of'251-asialoorosomucoid (ASOR) is linear for several hours at a rate of 15,000 molecules/cell/min. Neither the rate of'261-ASOR binding at 4" C nor the rate of uptake at 37" C was altered under conditions in which protein synthesis was completely inhibited. Thus each receptor recycles a ligand, on the average, every 16 min. The rate of binding of'251-ASOR to cells at 37" C, independent of internalization, was measured in the presence of 10-12 m~ azide. The rate of internalization at 37" C was determined from cells, prebound with ligand at 4" C, by following EDTA-resistant cellassociated"% ASOR. The distribution of functional re-ceptors within the cell was assessed from ligand binding at 4" C and ligand uptake at 37" C in cells pretreated with trypsin to remove all surface receptors. In growing Hep G2 cells 87% of the receptors are on the cell surface, and over 88% of these are unoccupied with ligand. During steady state internalization of'" 1-ASOR, the number of surface ASOR receptors decreases 17%. At 2 pg/ml of'261-ASOR, an unoccupied surface receptor requires a mean time of 8.7 min to bind a ligand, and a mean time of 2.2-2.3 min is required to internalize a surface receptor-ligand complex. An additional 4.2 min is required for ligand to dissociate from an internalized receptor and for the receptor to return to the cell surface. We have been able to determine or calculate most of these parameters of receptor recyclindby two or more independent sets of measurements.