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，使细胞质激酶结构域接近。这有助于酪氨酸的转磷酸化以稳定活性构象。虽然这个通用模型是有效的，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。这个受体酪氨酸激酶家族有5个细胞外免疫球蛋白样模块，一个跨膜（TM）螺旋，一个细胞内模块，由近膜结构域（JM），一个激酶结构域（激酶插入段）和一个c末端尾部组成。目的/假设：描述PDGFR-¿如何在膜上被抑制以及它如何在配体结合时被激活的分子细节是有限的。然而，PDGFR-¿的行为似乎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。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在质膜高度拥挤的环境中自发发生无意的转磷酸化和激活。研究设计：我计划研究PDGFR-¿的各个成分在其激活机制中的作用。在Aim 1中，我将研究细胞内模块在抑制和激活PDGFR-¿中的作用。我将生成删除JM域、c端尾部以及两者的结构。我将监测分离的激酶在溶液和脂质囊泡中的活性，以评估分子拥挤如何影响它们的功能。在Aim 2中，我将研究细胞外模块和TM螺旋在细胞中偶联配体结合激酶激活中的作用。我将研究在存在和不存在配体PDGF-BB的情况下的全长受体，以及两个缺失细胞外模块或在细胞外模块和TM螺旋之间插入连接器的突变受体。我将利用一种叫做双色脉冲交错激发荧光相互关联光谱（PIE-FCCS）的技术来评估这些变异如何影响受体的寡聚化，以及利用免疫荧光分析来评估细胞内激酶结构域的自磷酸化。在Aim 3中，我将评估质膜本身是否可以限制PDGFR-¿在细胞中的激活。使用Aim 2中描述的实验，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。健康相关性：PDGFR突变或过度表达引起的异常激活与许多人类癌症的发病机制有关。了解调控其激活的分子细节，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.