INVESTIGATION OF A NEWLY DISCOVERED ORGANELLE-BASED SIGNALING PARADIGM

项目概要/摘要 分子药理学的一个长期坚持的原则是，质膜是外部药物的唯一作用部位。 提示，如肽激素和生物胺，不能跨越这一障碍。这件事的核心 我的发现提出了一个建议，即一组长期以来被认为只从血浆中发出信号的受体 膜，也从亚细胞膜隔室发出信号。这种信号的划分 挑战了信号调节的一些基本范式。虽然现有数据已经显示， 区室化信号传导是许多膜受体的一般特征，本建议的重点是 β-肾上腺素能受体（β AR）是G蛋白偶联受体（GPCR）的一类。β AR控制强度， 心脏收缩频率和βAR信号紊乱是高血压和心力衰竭的基础。 β AR作为一种原型受体，其作用位点仅限于质膜， 受体从质膜上的去除通常被认为是 信令被终止。结合先进的成像平台与构象生物传感器， 我直接探测了βAR及其同源Gs蛋白的激活。我发现活跃的βAR-Gα 复合物不限于质膜，但也在亚细胞膜上积极地传递信号 这些细胞由内体和高尔基体组成。我进一步表明，不可渗透的激素，如 肾上腺素/去甲肾上腺素可以通过由一种有机化合物促进的机制到达高尔基体膜。 阳离子转运蛋白（OCT 3）。这些发现揭示了βAR的一个全新的调节成分 信号传导，即细胞内的区室化信号传导。我的长期目标是广泛了解 作为整合细胞生物学和信号传导的一种方式， 生理学和病理生理学。为了促进这一过程，我的实验室将重点研究βAR信号， 亚细胞膜隔室调节心脏功能。在本提案中，我们计划：1）阐明 βAR区室化信号传导在小鼠源性 2）阐明β AR在不同内环境下激活和失活的机制 3）阐明区室化信号传导在调节心输出量中的作用， 斑马鱼我们开发了使用纳米抗体的新工具来破坏特定受体/G蛋白偶联 膜定位，并将其与光遗传学方法相结合，以抑制区室化β AR 以剂量依赖性和可逆的方式进行信号传导。目前，GPCR是最受欢迎的 在药物靶点之后。大多数这些努力未能考虑到受体操纵在不同的亚细胞 分隔可以导致截然不同的结果，我们的初步研究已经暗示了这一概念。 问题研究因此，我们在理解区室化信号传导方面的努力有可能改变神经系统的功能。 用于开发与GPCR信号传导相关的有效小分子治疗剂的策略。

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.