THE EGF RECEPTOR--STRUCTURE, FUNCTION, AND HOMOLOGY

EGF受体底物磷酸化比活性为 增加了500倍(从每分钟2次的营业额增加到每分钟1000次 在30℃)在涉及双分子相互作用的变构过程中 受体分子和ATP的100多倍增长之间 公里。这让人想起了在不必要的和 F1-ATPase的多中心催化活性。仅含水泡 分离到非常高亲和力的EGF结合(Kd=0.1nM) 亲和力较低的膜(Kd=2-5 nm)与EGF结合。 这些囊泡中的受体被激活以获得多部位兴奋 比活性底物磷酸化活性。我们假设 EGF激活单分子受体自身磷酸化 启动受体内化的过程在特化的 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。这些实验 拟议遗嘱： 1.a.从费率的角度进一步定义多站点机制 需要高受体密度的确定过程，增加 ATP Km，克服了对ADP末端产物的强烈抑制； B.表征多肽的抑制特性，其可能 通过扰乱变构受体相互作用来发挥作用 多位底物磷酸化活性；以及 C.在磷脂囊泡中重建并表征 多位底物磷酸化催化体系； 2.a.确定EGF在其中起关键作用的关键反应 激活多位底物磷酸化； B.确定蛋白激酶C如何扰乱这一过程； 3.改进纯化亲和力极高的囊泡的程序 表皮生长因子受体对其多部位底物的研究 磷酸化特性；以及 4.尝试定义受体以外的成分 特异性受体内化的强制性导致 激活多位底物的磷酸化活性。 在另一项无关的研究中，EGF诱导酪氨酸磷酸化 培养细胞中的人糖皮质激素受体；这是 与糖皮质激素结合减少有关。纯净的 糖皮质激素受体将被纯化的EGF磷酸化 受体以检测配体结合活性是否降低。

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.