THE EGF RECEPTOR--STRUCTURE, FUNCTION, AND HOMOLOGY

EGF受体的底物磷酸化比活性是 增加了500倍（从2/min的周转次数增加到1000/min 在30 ℃下）在涉及双分子相互作用的变构过程中 受体分子与ATP增加100倍之间的联系 公里. 这让人想起了不确定性背后的过程， F1 ATP酶的多位点催化活性。 囊泡，仅 非常高亲和力的EGF结合（KD=0.1nM）被分离，不含 膜具有较低的亲和力（KD=2- 5 nM）EGF结合。 这些囊泡中的受体被激活， 底物磷酸化活性。 我们推测 EGF激活单分子受体自我磷酸化， 启动受体内化的过程，在一个专门的 一类具有高受体密度的囊泡，以支持 底物磷酸化的多位点模式。 实验 拟议将： 1. a. 在速率方面进一步定义多站点机制 决定过程需要高受体密度，增加 ATP Km，克服ADP终产物的强抑制; B. 表征多肽的抑制特性， 通过破坏所需的变构受体相互作用起作用 多位点底物磷酸化活性;和 C.在磷脂囊泡中重建并表征 催化多位点底物磷酸化的系统; 2. a. EGF在哪些方面起关键作用 激活多位点底物磷酸化; B. 定义蛋白激酶C如何破坏这一过程; 3. 用于纯化具有非常高亲和力的囊泡的改进程序 表皮生长因子受体，以表征其多位点底物 磷酸化性质;和 4. 尝试定义除受体以外的组件， 特异性受体内化的强制性， 激活多位点底物磷酸化活性。 在一项不相关的研究中，EGF诱导酪氨酸磷酸化， 人糖皮质激素受体在培养的细胞;这是 与糖皮质激素结合减少有关。 纯化 糖皮质激素受体将被纯化的EGF磷酸化 受体以测试降低的配体结合活性。

Substrate phosphorylation specific activity of EGF receptor is increased 500-fold (from a turnover number of 2/min to 1000/min at 30C) in an allosteric process involving bimolecular interactions between receptor molecules and an over 100-fold increase in ATP Km. This is reminiscent of the process underlying the unisite and multisite catalytic activities of F1 ATPase. Vesicles with only very high affinity EGF binding (KD=0.1nM) were isolated free of membranes with lower affinity (KD=2-5nM) EGF binding. Receptors in these vesicles are activated for multisite high specific activity substrate phosphorylation activity. We postulate that EGF activates a unimolecular receptor self-phosphorylation process that initiates receptor internalization in a specialized class of vesicles with high receptor density to support the multisite mode of substrate phosphorylation. The experiments proposed will: 1. a. Define the multisite mechanism further in terms of rate determining processes requiring high receptor density, increased ATP Km, and overcoming of strong ADP end product inhibition; b. Characterize inhibitory properties of polypeptides which may act by disrupting allosteric receptor interactions required for multisite substrate phosphorylation activity; and c. Reconstruct in phospholipid vesicles and characterize the system catalyzing multisite substrate phosphorylation; 2. a. Identify the pivotal reaction in which EGF is critical for activating multisite substrate phosphorylation; b. Define how protein kinase C disrupts this process; 3. Refine procedures for purifying vesicles with very high affinity EGF receptors to characterize their multisite substrate phosphorylation properties; and 4. Attempt to define components other than receptor which are obligatory for specific receptor internalization leading to activation of multisite substrate phosphorylation activity. In an unrelated study, EGF induced tyrosine phosphorylation of human glucocorticoid receptor in cultured cells; this was correlated with decreased glucocorticoid binding. Purified glucocorticoid receptor will be phosphorylated by purified EGF receptor to test for decreased ligand binding activity.