Effects of GPCR trafficking on the spatiotemporal control of signaling

项目总结/摘要 G蛋白偶联受体（GPCR）构成了一个信号受体家族，其基本上调节每一个信号转导通路。 生理过程最近的研究已经开始认识到G蛋白和GPCR在肿瘤中的作用 生物学特别是，β2-肾上腺素能受体（β2-AR）及其同源G蛋白突变的信号传导， GNAS与胰腺导管腺癌（PDAC）进展相关。了解 GPCR信号传导的基本分子机制对于实现其作为新的 治疗目标配体依赖性GPCR信号转导的研究传统上集中在受体 在质膜上活化。然而，最近发现细胞内区室也起作用， 作为引发不同信号结果的活性信号位点，提出了一个关键的生物学问题： GPCR运输决定了独特的信号，这些独特的信号会导致独特的细胞结果吗？的 建议的工作通过关注内体GPCR激活在形成cAMP中的作用来解决这个问题。 依赖的转录反应。首先，在已建立的HEK 293细胞模型中开始， 将评估贩运编码cAMP产生的数量和时间的机制。通过 受体和运输机制的操纵，内体停留时间和 将测试cAMP信号传导谱以确定运输是否足以形成cAMP输出或 是否需要其他因素，如翻译后修饰或相互作用蛋白。接下来 所提出的工作将定义这些不同的cAMP谱如何被解码以引起特定的下游反应。 将探测信号级联中的定义步骤，以确定转录应答是否 对cAMP产生的变化或细胞是否对cAMP刺激产生一致的反应敏感。 最后，拟议的研究将探讨HEK 293系统中描述的原则是否相关 对我们理解PDAC中β2-AR和GNAS的致癌信号传导有重要意义。时间、地点和持续时间 将评价胰腺导管上皮细胞中cAMP的产生以确定这些cAMP标记是否是 调节和后果以及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令人兴奋的领域-受体介导信号的时空控制。 这项研究还将为β2-AR的致癌信号传导机制提供新的见解， PDAC中的GNAS，以及更广泛地说，癌症中GPCR诱导的cAMP信号传导的范例。该项目将 在加州大学旧金山分校弗朗西斯科进行，由Mark von Zastrow博士指导， GPCR运输和信号传导领域的领导者，与胰腺癌领域的两位专家合作， 癌症（Dr. Eric Collisson，UCSF）和通过GPCR的致癌信号传导（Dr. J. Silvio Gutkind，UCSD）。此外 从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 具备能够成功开展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.