Effects of GPCR trafficking on the spatiotemporal control of signaling

项目摘要/摘要 G蛋白偶联受体(Gpcr)构成一个信号受体家族，基本上调节 生理过程。最近的研究已经开始认识到G蛋白和GPCRs在肿瘤中的作用 生物学。特别是，通过β2-肾上腺素能受体(β2-AR)及其同源G蛋白突变发出的信号， GNAS与胰腺导管腺癌(PDAC)的进展有关。了解 GPCR信号的基本分子机制将是实现其作为新的潜在功能的必要条件 治疗靶点。传统上，对配体依赖的gpr信号的研究主要集中在受体上。 质膜上的激活。然而，最近的发现，细胞内的隔室也发挥着作用 作为引起不同信号结果的活跃的信号位点，引发了一个关键的生物学问题：如何 GPCR的贩运决定了独特的信号，这些独特的信号是否会导致独特的细胞结果？这个 拟议的工作通过关注内体GPCR激活在形成cAMP-1中的作用来解决这个问题。 依赖转录反应。首先，从已建立的HEK293细胞模型开始，拟议的研究 将评估贩运活动编码产生营地的数量和时间的机制。穿过 受体和转运机制的操纵，内体滞留时间和转运机制的关系 将测试CAMP信号配置文件，以确定贩运是否足以塑造CAMP输出或 是否需要其他因素，如翻译后修饰或相互作用的蛋白质。接下来， 拟议的工作将定义如何对这些不同的阵营概况进行解码，以引起特定的下游反应。 将探索信号级联中定义的步骤，以确定转录反应 对cAMP生成的变化或细胞是否对cAMP刺激进化出统一的反应很敏感。 最后，拟议的研究将探讨HEK293系统中描述的原则是否相关 有助于我们理解β-2-AR和GNAS在肺泡巨噬细胞癌信号传导中的作用。时间、地点和持续时间 将评估胰腺导管上皮细胞中cAMP的生成，以确定这些cAMP信号是否 以及PDAC相关突变如何影响这种关系。这些研究将 推进基础细胞生物学的一个激动人心的领域--受体介导的信号的时空控制。 这项拟议的研究还将为β2-AR和BMP 2-AR的致癌信号机制提供新的见解。 在PDAC中的GNAS，以及更广泛地说，在癌症中GPCR诱导的cAMP信号的范例。这个项目将 在加州大学旧金山分校，在马克·冯·扎斯特罗博士的指导下进行 GPCR交易和信号领域的领导者，与胰腺领域的两名专家合作 癌症(Eric Collisson博士，加州大学旧金山分校)和GPCRs的致癌信号(J.Silvio Gutkind博士，加州大学伯克利分校)。此外 除了这些实验室的概念和实验培训，加州大学旧金山分校还提供了一个充满活力的研究社区 拥有能够成功实现拟议研究的专门知识和设施。

Project Summary/Abstract G protein coupled receptors (GPCRs) constitute a family of signaling receptors that regulate essentially every physiological process. Recent studies have begun to recognize the roles of G proteins and GPCRs in tumor biology. In particular, signaling by the β2-adrenergic receptor (β2-AR) and mutations in its cognate G protein, GNAS, have been associated with pancreatic ductal adenocarcinoma (PDAC) progression. Understanding the basic molecular mechanisms of GPCR signaling will be imperative for realizing their potential as novel therapeutic targets. Studies of ligand-dependent GPCR signaling have traditionally focused on receptor activation at the plasma membrane. However, the recent discovery that intracellular compartments also function as active signaling sites that elicit different signaling outcomes raises a critical biological question: how does GPCR trafficking dictate unique signals, and do these unique signals lead to unique cellular outcomes? The proposed work addresses this question by focusing on the role of endosomal GPCR activation in shaping cAMP- dependent transcriptional responses. First, starting in an established HEK293 cell model, the proposed studies will assess the mechanisms by which trafficking encodes the amount and timing of cAMP generation. Through manipulations of receptors and trafficking machinery, the relationship between endosomal residence time and cAMP signaling profiles will be tested to determine whether trafficking is sufficient to shape cAMP output or whether other factors, such as posttranslational modifications or interacting proteins, are required. Next, the proposed work will define how these distinct cAMP profiles are decoded to elicit a specific downstream response. Defined steps in the signaling cascade will be probed in order to ascertain whether the transcriptional response is sensitive to variations in cAMP generation or whether cells have evolved a uniform response to cAMP stimulus. Finally, the proposed studies will explore whether the principles delineated in the HEK293 system are relevant to our understanding of oncogenic signaling by β2-AR and GNAS in PDAC. The timing, location, and duration of cAMP generation in pancreatic ductal epithelial cells will be evaluated to determine if these cAMP signatures are regulated and consequential and how PDAC-associated mutations affect this relationship. These studies will advance an exciting area of fundamental cell biology—the spatiotemporal control of receptor-mediated signaling. The proposed research will also provide new insights into the mechanisms of oncogenic signaling by β2-AR and GNAS in PDAC and, more broadly, the paradigm of GPCR-induced cAMP signaling in cancer. This project will be carried out at the University of California, San Francisco under the mentorship of Dr. Mark von Zastrow, a leader in the field of GPCR trafficking and signaling, in collaboration with two experts in the fields of pancreatic cancer (Dr. Eric Collisson, UCSF) and oncogenic signaling by GPCRs (Dr. J. Silvio Gutkind, UCSD). In addition to the conceptual and experimental training from those labs, UCSF also offers a vibrant research community with expertise and facilities that will enable the successful realization of the proposed studies.