A431 cell variants lacking the blood group A antigen display increased high affinity epidermal growth factor-receptor number, protein-tyrosine kinase activity, and receptor turnover.
A431 cell variants lacking the blood group A antigen display increased high affinity epidermal growth factor-receptor number, protein-tyrosine kinase activity, and receptor turnover.
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缺乏血型A抗原显示的A431细胞变体增加了高亲和力表皮生长因子受体数，蛋白质酪氨酸激酶活性和受体更新。
DOI：
10.1083/jcb.107.3.939
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发表时间：
1988-09
影响因子：
7.8
通讯作者：
de Laat, S W
中科院分区：
文献类型：
--
作者：
Defize, L H;Arndt-Jovin, D J;Jovin, T M;Boonstra, J;Meisenhelder, J;Hunter, T;de Hey, H T;de Laat, S W
人A431细胞的表皮生长因子受体(EGF-R)具有与人A型血糖类结构密切相关的抗原决定簇。用A型血型反应性抗EGF-R单抗和各种凝集素标记研究表明，A431培养物在A型血型表达方面是异质性的。我们已经分离出这些细胞的克隆变体，它们要么表达(A431A+细胞)，要么完全缺乏A血型特异性N-乙酰-D-氨基半乳糖(GalNAc)残基。我们表明，这种差异是由于A431A-细胞中缺乏UDP-GalNAc：GAL转移酶活性所致。随后，我们比较了这些细胞系中EGF-R的功能。对EGF结合的Scatchard分析表明，在A431A-细胞中，6.3%的EGF-R属于高亲和力亚类(Kd=0.4 nM)，而在A431A+细胞中，这一亚类仅占总受体池的3.2%。A431A-细胞中高亲和力受体水平的升高伴随着受体蛋白酪氨酸激酶活性的平行增加。在A431A-细胞的膜制剂中，受体自磷酸化以及含有酪氨酸的多肽底物的磷酸化比A431A+细胞高2-3倍。在完整的A431A-细胞中，受体活性的差异被测量为受体磷酸化水平增加2-3倍，总细胞蛋白中磷酸酪氨酸的丰度增加2-3倍。此外，[35S]蛋氨酸脉冲追逐实验表明，A431A-细胞中EGF-R的翻转呈非配体依赖性增加：受体在这些细胞中的半衰期为10h，而在A431a+细胞中的半衰期为17h。我们的结果表明，GalNAc残基(S)可能参与了决定A431细胞中表皮生长因子受体亲和力、蛋白酪氨酸激酶活性和转化率的过程。此外，我们的结果表明，高亲和力的EGF-R是具有蛋白酪氨酸激酶活性的生物活性物种。
The epidermal growth factor receptor (EGF-R) of human A431 cells bears an antigenic determinant that is closely related to the human blood group A carbohydrate structure. Labeling studies with blood group A reactive anti-EGF-R monoclonal antibodies and various lectins revealed that A431 cultures are heterogeneous with respect to blood group A expression. We have isolated clonal variants of these cells that either express (A431A+ cells) or completely lack (A431A- cells) the blood group A specific N-acetyl-D-galactosamine (GalNAc) residue. We show that this difference is due to the absence of a UDP-GalNAc:Gal transferase activity in A431A- cells. Subsequently, we have compared EGF-R functioning in these cell lines. Scatchard analysis of EGF- binding shows that in A431A- cells 6.3% of the EGF-R belongs to a high affinity subclass (Kd = 0.4 nM) while in A431A+ this subclass represents only 3.2% of the total receptor pool. The elevated level of high affinity receptors in A431A- cells is accompanied by a parallel increase in receptor protein- tyrosine kinase activity. In membrane preparations of A431A- cells, receptor autophosphorylation as well as phosphorylation of a tyrosine-containing peptide substrate is 2-3-fold higher as compared with A431A+ cells. In intact A431A-cells, the difference in receptor activity is measured as a 2-3-fold elevated level of receptor phosphorylation and a 2-3-fold higher abundance of phosphotyrosine in total cellular protein in A431A- cells. In addition, [35S]methionine pulse-chase experiments showed a ligand-independent increase in turnover of EGF-R in A431A- cells: the receptor's half life in these cells is 10 h as compared with 17 h in A431A+ cells. Our results suggest a possible involvement of GalNAc residue(s) in determining EGF-R affinity, protein-tyrosine kinase activity and turnover in A431 cells. Furthermore, our results indicate that high affinity EGF-R are the biologically active species with respect to protein-tyrosine kinase activity.