Crystallization of Intact Receptor Tyrosine Kinases

描述(申请人提供)：受体酪氨酸激酶(RTK)，包括胰岛素和表皮生长因子(EGF)受体家族，在许多动物组织中调节细胞生长和分化。RTKs由一个胞外配体结合区、一个跨膜区和一个胞浆激酶组成。RTK的异常激活会导致细胞的异常生长，功能障碍的RTK与许多人类癌症的发生和严重程度有关，并成为许多抗癌药物的靶点：例如，在20-30%的人类乳腺癌中发现EGF受体同系物HER2的过度表达，并与更具侵袭性的肿瘤和不良预后相关，而抗HER2的单抗Herceptin已被证明是治疗这些癌症的有效药物。我的实验室最近开发了一种方法来生产高水平的可溶性富含半胱氨酸的糖蛋白，其中包括大多数RTK的胞外区域，以适合X射线结晶学分析的形式。这项工作导致了EGF受体胞外区的原子分辨结构，HER2，与Herceptin Fab络合的HER2，以及EGF受体同系物HER3。这些结构为配体结合如何产生信号提供了更多的见解，并启发了抗癌药物设计的新方法。然而，对于配体结合产生的信号是如何通过细胞膜传递的，人们几乎没有深入的了解。为了解决这个问题，需要完整受体的原子分辨结构。确定这些结构的一个主要障碍是以结晶形式表达和纯化足够数量的完整受体。我们的第一个目标是使我们开发的富含半胱氨酸的可溶性糖蛋白的表达方法适用于完整受体的表达和纯化。这些方法将普遍适用于所有细胞表面蛋白的结构研究。我们的第二个目标是应用这些方法来过度表达EGF和胰岛素受体家族的成员。我们的第三个目标是提纯和鉴定这些受体。我们的最终目标是生产这些受体的衍射级晶体，既有单独的，也有与配体络合的。

DESCRIPTION (provided by applicant): Receptor tyrosine kinases (RTKs), which include the insulin and epidermal growth factor (EGF) receptor families, mediate cell growth and differentiation in many animal tissues. RTKs consist of an extracellular ligand binding region, a single transmembrane spanning region, and a cytoplasmic kinase. Inappropriate activation of RTKs results in abnormal cell growth, and dysfunctional RTKs have been implicated in the genesis and severity of many human cancers and become the target of many anticancer drugs: For example, overexpression of the EGF receptor homolog HER2 is found in 20-30% of human breast cancers and correlates with more aggressive tumors and a poorer prognosis, and an anti-HER2 monoclonal antibody, Herceptin, has proven an effective treatment for these cancers. My laboratory has recently developed an approach to producing high levels of soluble cysteine-rich glycoproteins, which includes the extracellular regions of most RTKs, in forms suitable for X-ray crystallographic analysis. This work has led to atomic resolution structures of extracellular regions of the EGF receptor, HER2, HER2 complexed with the Herceptin Fab, and the EGF receptor homolog HER3. These structures have provided much insight into how ligand binding generates signals and inspired new approaches to anticancer drug design. Little insight has been gained, however, into how the signal produced by ligand binding is transduced across the cell membrane. Atomic resolution structures of intact receptors are needed to solve this problem. A major roadblock to determination of these structures is the expression and purification of sufficient amounts of intact receptors in crystallizable form. Our first aim is to adapt the expression methods that we have developed for soluble cysteine-rich glycoproteins to the expression and purification of intact receptors. These methods will be generally applicable to structural studies of all cell surface proteins. Our second aim is to apply these methods to overexpress members of the EGF and insulin receptor families. Our third aim is to purify and characterize these receptors. Our final aim is to produce diffraction-quality crystals of these receptors both alone and complexed with ligand.