INVESTIGATION OF A NEWLY DISCOVERED ORGANELLE-BASED SIGNALING PARADIGM

项目摘要/摘要 分子药理学的一个长期信条是质膜是外源药物的唯一作用部位。 提示，如肽激素和生物胺，不能越过这一障碍。这件事的核心是 我的发现提出了一种建议，即长期以来被认为只从血浆中发出信号的一组受体 膜，也是来自亚细胞膜室的信号。这种信令的划分 挑战信号调控的一些基本范式。虽然现有的数据已经表明 信号的区隔是许多膜受体的一个普遍特征，本提案的重点是 β肾上腺素能受体(βARs)是G蛋白偶联受体(GPCRs)的一类。βAR控制着力量和力量 心脏收缩和βAR信号紊乱的频率是高血压和心力衰竭的基础。 β受体是一种典型的受体，其作用部位仅限于质膜和 从质膜上去除受体通常被认为是 信令终止。将复杂的成像平台与构象生物传感器相结合，即 发展起来，我直接探测了βAR及其同源Gs蛋白的激活。我发现ActiveβAR-GαS 复合体不仅限于质膜，而且在亚细胞膜上也是活跃的信号 内小体和高尔基体等隔室。我进一步证明，不透水的荷尔蒙 肾上腺素/去甲肾上腺素可通过一种由有机物质促进的机制到达高尔基体膜 阳离子转运蛋白(10月3日)。这些发现揭示了βAR的一个全新的监管组成部分 信令，即细胞内的信令，被划分为不同的信令。我的长期目标是广泛地理解 将细胞生物学和信号转导作为一种整合方式的功能后果 生理学和病理生理学。为了促进这一过程，我的实验室将专注于βAR信令 亚细胞膜室对心脏功能的调节作用。在这项提议中，我们计划：1)阐明 小鼠脑内β-AR信号区划的分子和细胞后果 心肌细胞，2)阐明不同内源性β受体激活和失活的机制 膜间隔3)阐明了间隔信号在调节心输出量中的作用。 斑马鱼。我们已经开发出新的工具，使用纳米抗体，在特定的位置干扰受体/G蛋白偶联 膜的位置，并将它们与光遗传方法相结合来抑制区域化的β受体 以剂量依赖和可逆的方式发出信号。目前，GPCR是最受欢迎的 追查毒品目标。大多数这些努力都没有考虑到不同亚细胞的受体操纵 分舱可能会导致截然不同的结果，这一概念已经在我们的初步研究中得到了暗示 学习。因此，我们在理解划分的信令方面的努力有可能改变 用于开发与gpr信号相关的有效小分子疗法的策略。

Project Summary/Abstract A long-held tenet of molecular pharmacology is that the plasma membrane is the sole site of action of external cues, such as peptide hormones and biogenic amines, which cannot cross this barrier. The centerpiece of this proposal emerges from my discoveries that a set of receptors long considered to only signal from the plasma membrane, also signal from subcellular membrane compartments. This compartmentalization of signaling challenges some of the basic paradigms of signaling regulation. While the existing data already shows that compartmentalized signaling is a general feature of many membrane receptors, the focus of this proposal is on β-adrenergic receptors (βARs) class of G Protein Coupled Receptors (GPCRs). βARs control the strength and frequency of cardiac contraction and disturbances in βAR signaling underlie hypertension and heart failure. βARs have served as a prototypical receptor whose sites of action are limited to plasma membrane and removal of the receptors from the plasma membrane has been generally viewed as the mechanism by which signaling is terminated. Combining sophisticated imaging platforms with conformational biosensor that I developed, I directly probed activation of βAR and its cognate Gs protein. I discovered that active βAR-Gαs complex is not restricted to the plasma membrane but is also actively signals at subcellular membrane compartments such as endosomes and the Golgi. I further showed that impermeable hormones such as epinephrine/norepinephrine can reach the Golgi membranes through a mechanism facilitated by an organic cation transporter (OCT3). These findings have uncovered an entirely new regulatory component to βAR signaling, namely intracellular, compartmentalized signaling. My long-term goal is to broadly understand the functional consequence of compartmentalized signaling as a way of integrating cell biology and signaling with physiology and pathophysiology. To facilitate this process, my laboratory will focus on βAR signaling from subcellular membrane compartment in regulating cardiac functions. In this proposal we plan to: 1) Elucidate the molecular and cellular consequences of βAR compartmentalized signaling in mouse-derived cardiomyocytes, 2) Elucidate the mechanism of activation and inactivation of βARs at different internal membrane compartments 3) Elucidate the role of compartmentalized signaling in regulating cardiac outputs in zebrafish. We have developed new tools, using nanobodies, to disrupt receptor/G protein coupling at specific membrane locations and are combining them with an optogenetic approach to inhibit compartmentalized βARs signaling in a dose dependent and reversible manner. Currently, GPCRs are among the most heavily sought after drug targets. Most of these efforts fail to consider that receptor manipulation at different subcellular compartments can cause vastly different outcomes, a notion that is already hinted at by our preliminary studies. Thus, our efforts at understanding compartmentalized signaling has the potential of transforming the strategies that are used for developing effective small molecule therapeutics that link to GPCR signaling.