CROSS-LINKING OF EPIDERMAL GROWTH-FACTOR RECEPTORS IN INTACT-CELLS - DETECTION OF INITIAL-STAGES OF RECEPTOR CLUSTERING AND DETERMINATION OF MOLECULAR-WEIGHT OF HIGH-AFFINITY RECEPTORS
CROSS-LINKING OF EPIDERMAL GROWTH-FACTOR RECEPTORS IN INTACT-CELLS - DETECTION OF INITIAL-STAGES OF RECEPTOR CLUSTERING AND DETERMINATION OF MOLECULAR-WEIGHT OF HIGH-AFFINITY RECEPTORS
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DOI：
10.1021/bi00369a011
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发表时间：
1986-10-21
期刊：
影响因子：
2.9
通讯作者：
CIDLOWSKI, JA
中科院分区：
文献类型：
--
作者：
FANGER, BO;AUSTIN, KS;CIDLOWSKI, JA
本文报道了一种用化学交联法用~（125）I-EGF标记全细胞表皮生长因子（EGF）受体的方法。聚丙烯酰胺凝胶电泳分离出一个Mr. apprx。180，000 EGF-受体复合物和更大的Mr. 360 000个聚合物。较大的复合物的形成是时间和温度依赖性的，似乎代表了EGF受体簇的初始事件。125 I-EGF标记的高-（Kd apprx. 0.16 nM）和低-（Kd. 1.5通过与未标记的EGF竞争或通过用地塞米松诱导另外的高亲和力位点，表明两个位点都由Mr. apprx代表。180，000个125 I-EGF受体复合物。在交联之前消化细胞检测到一小群胰蛋白酶抗性的Mr. apprx。180，000个受体，其可以代表先前描述的隐蔽和/或高亲和力受体。几个. apprx先生。360，000个受体是胰蛋白酶抗性的。高亲和力EGF受体的糖皮质激素诱导未能诱导EGF受体微簇的可检测变化，但确实导致HeLa S3细胞膜中EGF诱导的受体磷酸化在4 ° C下增加50%。C.因此，糖皮质激素增加高亲和力EGF结合位点，EGF诱导的受体磷酸化和细胞生长。
A method was developed to label epidermal growth factor (EGF) receptors with 125I-EGF in whole cells using chemical cross-linking reagents. Polyacrylamide gel electrophoresis resolved an Mr .apprx. 180,000 EGF-receptor complex and larger Mr .gtoreq. 360,000 aggregates. The formation of the larger complexes was time and temperature dependent and appeared to represent the initial events of EGF receptor clustering. Alteration of the ratio of 125I-EGF-labeled high- (Kd .apprx. 0.16 nM) and low- (Kd .apprx. 1.5 nM) affinity complexes by competition with unlabeled EGF or by induction of additional high -affinity sites with dexamethasone suggested that both sites were represented by the Mr .apprx. 180,000 125I-EGF-receptor complexes. Digestion of cells before cross-linking detected a small population of trypsin-resistant Mr .apprx. 180,000 receptors, which could represent previously described cryptic and/or high-affinity receptors. Few of the Mr .apprx. 360,000 receptor were trypsin resistant. Glucocorticoid induction of high-affinity EGF receptors failed to induce detectable changes in the microclustering of EGF receptors but did result in a 50% increase in EGF-induced receptor phosphorylation in HeLa S3 cell membranes at 4.degree. C. Thus, glucocorticoids increase high-affinity EGF binding sites, EGF-induced receptor phosphorylation, and cell growth.