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

描述（由申请人提供）：受体酪氨酸激酶激活尚不完全清楚。在经典模型中，配体诱导两个受体的细胞外模块的二聚化以使细胞质激酶结构域紧密接近。这促进酪氨酸的转磷酸化以稳定活性构象。虽然这种一般模式是有效的，但还存在许多额外的监管层。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。具体来说，限制PDGFR在膜上激活的详细机制是有争议的。该受体酪氨酸激酶家族具有五个细胞外免疫球蛋白样模块、跨膜（TM）螺旋和由跨膜结构域（JM）、具有激酶插入片段的激酶结构域和C末端尾组成的细胞内模块。目的/假设：描述PDGFR-β如何在膜上被抑制以及它如何在配体结合后被激活的分子细节是有限的。然而，PDGFR-<$的行为似乎与受体酪氨酸激酶活化的经典模型不同。因此，我计划系统地研究各个域对PDGFR-β的调节作用。该系统的分子表征将提供深入了解受体酪氨酸激酶如何防止意外的转磷酸化和激活自发发生在高度拥挤的环境中的质膜。研究设计：我计划研究PDGFR-<$的单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-β中的作用。我将生成删除JM结构域、C末端尾以及两者的构建体。我将监测分离的激酶在溶液中和脂质囊泡上的活性，以评估分子拥挤如何影响它们的功能。在目的2中，我将研究细胞外模块和TM螺旋在偶联配体结合到细胞中激酶活化中的作用。我将研究全长受体的存在和不存在的配体，PDGF-BB，以及两个突变受体与细胞外模块删除或连接器插入之间的细胞外模块和TM螺旋。我将评估这些变体如何影响使用称为双色脉冲交错激发荧光互相关光谱（PIE-FCCS）的技术的受体的寡聚化和使用免疫荧光测定的细胞内激酶结构域的自磷酸化。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中描述的测定，我将研究当膜以定位基序靶向质膜时，膜仅对受体的细胞内模块的影响。健康危害：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如何影响受体，并可能导致新的治疗方法。

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.