Directed Evolution of Cell-Type Specific On-Demand Signaling Control Systems

项目总结/摘要 这个项目将扩展我最近在定向进化方面的发明，以创造细胞类型特异性的工具。 G蛋白偶联受体的光开关调谐。人类基因组编码>900个G蛋白 偶联受体（GPCR）。GPCR存在于每一个细胞中，参与每一个已知的生物过程; 从我们的味觉、嗅觉和视觉，到协调激素、神经递质和免疫系统， 功能协调发展的过去一百年来对这个蛋白质家族的生物化学研究已经产生了 346个结构测定，> 100，000个已确认的配体，以及丰富的信号分析 平台在这份令人印象深刻的名单中，没有任何用于离散的、特定于细胞类型的调优的工具 内源性受体活性（图1）。没有这样的工具，我们就缺乏直接连接的能力 离散的GPCR信号传导事件转化为细胞类型特异性生理输出。这些工具至关重要， 我们将了解GPCR如何决定人类健康和疾病。在这个NIH创新者 我将扩大我最近发明的哺乳动物定向进化平台， GPCR的细胞类型特异性、光可切换变构调节剂的生产线。然后我将 开发第一个D1和D2多巴胺GPCR的调节剂，并使用这些工具来完善 小鼠大脑中多巴胺能奖励信号的现有模型。这项提案的成功 将有利于生物医学研究的每一个领域，创造一个管道，以获得可调的控制，不仅 任何GPCR，但任何细胞信号蛋白。

Project Summary/Abstract This project will expand my recent inventions in directed evolution to create tools for cell-type specific photoswitchable tuning of G-protein coupled receptors. The human genome encodes >900 G-protein coupled receptors (GPCRs). Found in every cell, GPCRs contribute to every known biological process; from our sense of taste, smell, and sight to coordinating hormone, neurotransmitter, and immune functions. The past one-hundred years of biochemical research on this protein family has produced 346 structural determinations, >100,000 confirmed ligands, and a wealth of signaling assay platforms. Absent from this impressive roster are any tools for the discrete, cell-type specific tuning of endogenous receptor activity (Fig 1). Without such tools, we lack the ability to directly connect discrete GPCR signaling events to cell-type specific physiological outputs. Such tools are essential if we are to understand how GPCRs dictate human health and disease. In this NIH New Innovator proposal, I will expand my recently invented platform for mammalian directed evolution to create a production pipeline for cell-type specific, photoswitchable allosteric modulators of GPCRs. I will then develop the first of these modulators for the D1 and D2 dopamine GPCRs and use these tools to refine existing models of dopaminergic reward signaling in the murine brain. The success of this proposal will benefit every field of biomedical research, creating a pipeline to gain tunable control of not only any GPCR, but any cell signaling protein.