Elucidating the mechanism of PDGFR- B activation at the plasma membrane

描述(由申请人提供)：受体酪氨酸激酶的激活尚不完全清楚。在经典的模型中，一个配体诱导两个受体的胞外模块二聚化，使胞浆内的激酶域非常接近。这有助于酪氨酸的转磷酸化以稳定活性构象。虽然这一一般模型是有效的，但还有许多额外的监管层面。最近的研究表明，不同的受体酪氨酸激酶家族可能有不同的调节模式。具体地说，限制PDGFR在膜上激活的详细机制存在争议。这个受体酪氨酸激酶家族有五个细胞外免疫球蛋白样模块、一个跨膜(TM)螺旋和一个细胞内模块，该模块包括一个膜旁结构域(JM)、一个带有激酶插入片段的激动域和一个C-末端尾巴。目的/假设：描述PDGFR如何在膜上被抑制以及如何在配体结合时被激活的分子细节是有限的。然而，PDGFR-？似乎并不符合受体酪氨酸激酶激活的经典模型。因此，我计划系统地研究各个结构域对PDGFR-？的调节作用。对这一系统的分子表征将提供关于受体酪氨酸激酶如何防止在高度拥挤的质膜环境中无意中发生转磷酸化和激活的深入了解。研究设计：我计划研究PDGFR-1的各个成分在其激活机制中的作用。在目标1中，我将研究细胞内模块在抑制和激活PDGFR-1中的作用。我将生成删除JM结构域、C-末端尾部和两者的构建。我将监测分离的激酶在溶液中和脂泡上的活性，以评估分子拥挤如何影响它们的功能。在目标2中，我将研究细胞外模块和TH TM螺旋在细胞内偶联配体结合到激酶激活中的作用。我将研究在配体PDGF-BB存在和不存在的情况下的全长受体，以及两个突变的受体，这些受体要么缺失胞外模块，要么在胞外模块和TM螺旋之间插入连接物。我将评估这些变体如何影响使用双色脉冲交错激发荧光互相关光谱(PIE-FCCS)技术的受体寡聚和使用免疫荧光分析的细胞内激酶域的自动磷酸化。在目标3中，我将评估质膜本身是否可以限制细胞中PDGFR的激活。使用目标2中描述的分析方法，我将研究当膜针对具有定位基序的质膜时，膜对受体的细胞内模块的影响。与健康相关：由于突变或过度表达导致的PDGFR异常激活与许多人类癌症的发病机制有关。了解调节其激活的分子细节可以深入了解致癌突变是如何影响受体的，并可以导致新的治疗方法。

DESCRIPTION (provided by applicant): Receptor tyrosine kinase activation is incompletely understood. In the classic model, a ligand induces dimerization of the extracellular modules of two receptors to bring the cytoplasmic kinase domains into close proximity. This facilitates transphosphorylation of tyrosines to stabilize an active conformation. While this general model is valid, many additional layers of regulation exist. Recent studies indicate that different receptor tyrosine kinase families may have distinct modes of regulation. Specifically, the detailed mechanism that restricts PDGFR activation at the membrane is controversial. This family of receptor tyrosine kinases has five extracellular immunoglobulin-like modules, a transmembrane (TM) helix, and an intracellular module that consists of a juxtamembrane domain (JM), a kinase domain with a kinase insert segment, and a C-terminal tail. OBJECTIVE/HYPOTHESIS: The molecular details that depict how PDGFR-¿ is inhibited at the membrane and how it becomes activated upon ligand binding are limited. However, PDGFR-¿ does not appear to behave according to the classic model for receptor tyrosine kinase activation. Thus, I plan to systematically investigate the regulatory roles of the individual domains on PDGFR-¿. Molecular characterization of this system will provide insight into how receptor tyrosine kinases prevent inadvertent transphosphorylation and activation from occurring spontaneously in the highly crowded environment at the plasma membrane. STUDY DESIGN: I plan to investigate the role of the individual components of PDGFR-¿ in its activation mechanism. In Aim 1, I will investigate the role of the intracellular module in inhibiting and activating PDGFR-¿. I will generate constructs with deletion of the JM domain, the C-terminal tail, and both. I will monitor the activiy of the isolated kinases in solution and on lipid vesicles to evaluate how molecular crowding influences their function. In Aim 2, I will investigate the role of the extracellular module and th TM helix in coupling ligand binding to kinase activation in cells. I will study the full-length receptor in the presence and absence of ligand, PDGF-BB, as well as two mutant receptors with either the extracellular module deleted or a linker inserted between the extracellular module and the TM helix. I will evaluate how these variants influence both oligomerization of the receptor using a technique called two-color pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS) and autophosphorylation of the intracellular kinase domain using immunofluorescence assays. In Aim 3, I will evaluate if the plasma membrane itself can restrict PDGFR-¿ activation in cells. Using the assays described in Aim 2, I will study the effect that the membrane has on just the intracellular module of the receptor when it is targeted to the plasma membrane with a localization motif. HEALTH RELATEDNESS: Abnormal activation of PDGFR due to mutation or overexpression is implicated in the pathogenesis of many human cancers. Understanding the molecular details that regulate its activation can provide insight into how oncogenic mutations affect the receptor and can lead to novel therapeutic approaches.