Ben-Levy R, Peles E, Goldman-Michael R, Yarden Y
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
J Biol Chem. 1992 Aug 25;267(24):17304-13.
The neu protooncogene encodes a receptor tyrosine kinase homologous to the receptor for the epidermal growth factor. The oncogenic potential of neu is released upon chemical carcinogenesis, which replaces a glutamic acid for a valine residue, within the single transmembrane domain. This results in constitutive receptor dimerization and activation of the intrinsic catalytic function. To study the implications of the oncogenic mutation and the consequent receptor dimerization on the interaction with the yet incompletely characterized ligand of p185neu, we constructed chimeric proteins between the ligand binding domain of the epidermal growth factor receptor and the transmembrane and cytoplasmic domains of the normal or the transforming Neu proteins. The chimeric receptors displayed cellular and biochemical differences characteristic of the normal and the transforming Neu proteins and therefore may reliably represent the ligand binding functions of the two receptor forms. Analyses of ligand binding revealed qualitative and quantitative differences that were a result of the single mutation; whereas the normal chimera (valine version) displayed two populations of binding sites with approximately 90% of the receptors in the low affinity state, the transforming receptor (glutamic acid version) showed a single population of binding sites with relatively high affinity. Kinetics measurements indicated that the difference in affinities was because of slower rates of both ligand association and ligand dissociation from the constitutively dimerized mutant receptor. It therefore appears that the oncogenic mutation, by permanently dimerizing the receptor, establishes a high affinity ligand binding state which is functionally equivalent to the ligand-occupied normal receptor. Our conclusion is further supported by the rates of endocytosis of the wild-type and the mutant receptor. Hence, these results provide the first experimental evidence from living cells which supports a model that attributes the heterogeneity of ligand binding sites to the state of oligomerization of receptor tyrosine kinases.
神经原癌基因编码一种与表皮生长因子受体同源的受体酪氨酸激酶。在化学致癌过程中，神经原癌基因的致癌潜能被释放，此时在单个跨膜结构域内，一个缬氨酸残基被谷氨酸取代。这导致受体组成型二聚化并激活内在催化功能。为了研究致癌突变以及由此产生的受体二聚化对与尚未完全表征的p185neu配体相互作用的影响，我们构建了表皮生长因子受体配体结合结构域与正常或转化型Neu蛋白的跨膜和胞质结构域之间的嵌合蛋白。嵌合受体表现出正常和转化型Neu蛋白特有的细胞和生化差异，因此可以可靠地代表两种受体形式的配体结合功能。配体结合分析揭示了由单一突变导致的定性和定量差异；正常嵌合体（缬氨酸版本）显示出两种结合位点群体，约90%的受体处于低亲和力状态，而转化型受体（谷氨酸版本）显示出单一群体的具有相对高亲和力的结合位点。动力学测量表明，亲和力的差异是由于配体与组成型二聚化突变受体的结合和解离速率都较慢。因此，致癌突变通过使受体永久二聚化，似乎建立了一种高亲和力配体结合状态，其功能等同于配体占据的正常受体。野生型和突变型受体的内吞速率进一步支持了我们的结论。因此，这些结果提供了来自活细胞的首个实验证据，支持了一种将配体结合位点的异质性归因于受体酪氨酸激酶寡聚化状态的模型。
