Activation of phospholipase C beta enzymes by G beta-gamma and corresponding regulation of downstream ion channels

项目摘要 细胞通过G蛋白偶联受体（GPCR）对许多细胞外刺激作出反应。细胞外信号传导 分子结合GPCR并催化细胞内膜锚定的G蛋白Ga和Gbg的释放， 作用于下游目标。离子通道是许多GPCR信号级联的下游靶标， 将细胞对这些刺激的反应与膜兴奋性联系起来。依赖GPCR的离子调节 通道在包括伤害感受，心率调节， 和炎症;因此，了解这种调节是必要的。离子通道可通过GPCR调节 信号直接通过G蛋白或G蛋白调节的第二信使，包括磷脂酰肌醇， 4，5-二磷酸（PIP2）。PIP 2被磷脂酶C（PLC）的G蛋白依赖性B家族降解 酶，其裂解PIP2以产生IP3和DAG。许多离子通道家族受PIP2调节 以PLC b依赖的方式，包括内向整流K+（Kir）通道和电压依赖性K+通道。 PLCb酶被Gaq和Gbg激活，将其功能与Gaq和Gai偶联受体连接。 虽然Gaq的调节是很好理解的，但关于Gbg依赖性激活还有很多未知之处。在 为了了解下游离子通道的GPCR依赖性调节和相关的 生理过程，有必要了解PLCb酶的G蛋白调节，这是 关键信号中间体。为此，我建议通过以下方式研究PLCb酶的Gbg依赖性激活： 以下两个目的：（1）研究PLCb酶的Gbg依赖性活化的最低要求 和使用无细胞重构系统调节下游离子通道，（2）表征 Gbg和PLCb之间的相互作用，包括结合位点的定位和Gbg的表征。 用低温电子显微镜和生物发光共振技术研究依赖性构象变化 能量转移这些目标的成功完成将直接连接PLCb调节其对离子的影响 通道，扩展这些信号级联的知识。该项目将在 洛克菲勒大学的罗德里克·麦金农博士的指导。麦金农博士有丰富的经验 研究离子通道的功能和调节，培养博士后研究人员， 独立研究员。实验室和洛克菲勒大学共同创造了一个环境， 完成拟议项目所需的资源和智力投入。配套培训计划 包括一个时间轴，描述所提出的实验的完成情况，并为个人和 职业发展，包括经常与麦金农博士会面，参加会议，介绍 研究结果，指导学生，并授予写作，以获得独立研究计划的资金。

Project Summary Cells respond to many extracellular stimuli via G protein coupled receptors (GPCR). Extracellular signaling molecules bind GPCR’s and catalyze the release of intracellular membrane anchored G proteins, Ga and Gbg, which act on downstream targets. Ion channels are a downstream target of numerous GPCR signaling cascades, connecting the cellular response to these stimuli to membrane excitability. GPCR-dependent regulation of ion channels plays an essential role in many physiological processes including nociception, regulation of heart rate, and inflammation; therefore, it is essential to understand this regulation. Ion channels can be regulated by GPCR signaling directly by G proteins or by G protein-regulated second messengers, including phosphatidylinositol- 4,5-bisphosphate (PIP2). PIP2 is degraded by the G protein-dependent b family of phospholipase C (PLC) enzymes, which cleave PIP2 to produce IP3 and DAG. Numerous families of ion channels are regulated by PIP2 in a PLCb-dependent manner, including inwardly rectifying K+ (Kir) channels and voltage-dependent K+ channels. PLCb enzymes are activated by both Gaq and Gbg, linking their function to both Gaq and Gai-coupled receptors. While the regulation by Gaq is well-understood, much is unknown regarding the Gbg-dependent activation. In order to understand the GPCR-dependent regulation of downstream ion channels and the associated physiological processes, it is necessary to understand the G protein regulation of PLCb enzymes, which are the key signaling intermediate. To this end, I propose to study the Gbg-dependent activation of PLCb enzymes via the following two aims: (1) Investigate the minimal requirements for Gbg-dependent activation of PLCb enzymes and the regulation of downstream ion channels using a cell-free reconstituted system, (2) Characterize the interaction between Gbg and PLCb including localization of the binding site and characterization of the Gbg- dependent conformational changes using cryogenic electron microscopy and bioluminescence resonance energy transfer. Successful completion of these aims will directly connect PLCb regulation to its effect on ion channels, expanding the knowledge of these signaling cascades. This project will be conducted under the supervision of Dr. Roderick MacKinnon at Rockefeller University. Dr. MacKinnon has extensive experience studying the function and regulation of ion channels as well as training postdoctoral researchers to succeed as independent researchers. Together, the lab and Rockefeller University generate an environment with the resources and intellectual input necessary for completing the proposed project. The accompanying training plan includes a timeline describing the completion of the proposed experiments and allocates time for personal and career development including frequent meetings with Dr. MacKinnon, attending conferences to present the findings, mentoring students, and grant writing to acquire funding for an independent research program.