Quantitative analysis of the endocytic system involved in hormone-induced receptor internalization.
Quantitative analysis of the endocytic system involved in hormone-induced receptor internalization.
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发表时间：
1990-09
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通讯作者：
K. Lund;L. Opresko;C. Starbuck;B. Walsh;H. Wiley
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作者：
K. Lund;L. Opresko;C. Starbuck;B. Walsh;H. Wiley
我们已经开发了一种定量方法来评估细胞表面受体和内吞装置之间的相互作用。这种方法利用了在表达大量受体的细胞中发生的内化率的占用依赖性变化。我们发现转铁蛋白受体的组成性内化表现为一个简单的一级过程，不受配体的影响。然而，表皮生长因子（EGF）受体的内化表现为由受体占据诱导的可饱和的二级过程。EGF受体的内化通过至少两种不同的途径发生：对被占据的受体具有相对高亲和力的低容量途径和具有高得多的容量的低亲和力途径。在所有具有内在酪氨酸激酶活性受体的细胞中观察到高亲和力途径。缺乏激酶活性的突变型EGF受体不能利用高亲和力途径，只能通过低亲和力途径内化。对激酶底物亲和力降低的突变受体也通过高亲和力、诱导途径以降低的速率内化。在非洲爪蟾卵母细胞中的卵黄蛋白原受体的情况下，占用的受体竞争更有效地比空的内化。胰岛素可增加卵母细胞对卵黄蛋白原受体的内吞能力。同样，血清增加了哺乳动物细胞中EGF受体诱导途径的能力。这些数据与内化模型一致，其中占据的受体结合到介导快速内化的特定细胞组分。配体诱导的内化是由于被占据的受体对内吞器官的亲和力增加所致。激素也可以通过增加被膜小凹的数量或它们的内化速率来间接调节内吞作用。将受体特异性效应与细胞特异性效应区分开来，对于研究受体介导的内吞作用的分子机制是非常有用的。
We have developed a quantitative method to evaluate the interaction between cell surface receptors and the endocytic apparatus. This method exploits occupancy-dependent changes in internalization rates that occur in cells expressing high numbers of receptors. We found that constitutive internalization of the transferrin receptor behaves as a simple, first order process that is unaltered by ligand. Internalization of the epidermal growth factor (EGF) receptor, however, behaves as a saturable, second order process that is induced by receptor occupancy. Internalization of EGF receptors occurs through at least two distinct pathways: a low capacity pathway that has a relatively high affinity for occupied receptors, and a low affinity pathway that has a much higher capacity. The high affinity pathway was observed in all cells having receptors with intrinsic tyrosine kinase activity. Mutant EGF receptors lacking kinase activity could not utilize the high affinity pathway and were internalized only through the low affinity one. Mutated receptors with decreased affinity for kinase substrates were also internalized at decreased rates through the high affinity, inducible pathway. In the case of vitellogenin receptors in Xenopus oocytes, occupied receptors competed more efficiently for internalization than empty ones. Insulin increased the endocytic capacity of oocytes for vitellogenin receptors. Similarly, serum increased the capacity of the inducible pathway for EGF receptors in mammalian cells. These data are consistent with a model of internalization in which occupied receptors bind to specific cellular components that mediate rapid internalization. Ligand-induced internalization results from an increase in the affinity of occupied receptors for the endocytic apparatus. Hormones can also indirectly regulate endocytosis by increasing the number of coated pits or their rate of internalization. The ability to dissect receptor-specific effects from cell-specific ones should be very useful in investigating the molecular mechanisms of receptor mediated endocytosis.