Molecular mechanisms of proton-sensing in CO2-dependent breathing

摘要 GPR4是一种G蛋白偶联受体，是二氧化碳依赖呼吸所必需的。在脑干中，上升的二氧化碳 水平会导致pH降低，从而激活GPR4。GPR4基因敲除导致两种病理 表型-减少对二氧化碳的依赖呼吸和频繁呼吸暂停，这两种情况都可以通过特定的 GPR4在脑干中的重新表达。这在GPR4失活和症状之间建立了联系 治疗严重的呼吸障碍，包括先天性中枢性低通气综合征(CCHS)和中枢睡眠 呼吸暂停(CsA)。然而，我们不知道GPR4是如何感知pH的，我们也不知道GPR4如何存在于 分子调节GPR4。通过这一提议，我将确定GPR4的分子机制 激活，以提供对二氧化碳依赖呼吸的调节的见解，并使发展 激活GPR4的小分子。在目标1中，我将确定质子如何激活的分子细节 GPR4。在目标2中，我将破译当前的拮抗者如何结合和无效的GPR4。为了达到这些目标，我 将结合生物化学、结构生物学(低温电子显微镜)和 药理学(诱变和信号分析)。这些研究将提供对 通过质子和小分子调节GPR4活性。这将扩大我们的根本 理解质子如何驱动二氧化碳依赖的呼吸，这将使未来能够 激活GPR4的小分子，可能成为治疗CCHS和CSA的新药物。

Abstract GPR4 is a G-protein coupled receptor required for CO2-dependent breathing. In the brainstem, rising CO2 levels cause a decrease in pH that activates GPR4. Genetic knockout of GPR4 results in two pathological phenotypes - reduced CO2-dependent breathing and frequent apneas, which are both rescued by specific re-expression of GPR4 in the brainstem. This establishes a link between inactivation of GPR4 and symptoms for severe breathing disorders including congenital central hypoventilation syndrome (CCHS) and central sleep apnea (CSA). However we do not know how GPR4 senses pH, and we do not know how existing small molecules modulate GPR4. Through this proposal, I will determine the molecular mechanism of GPR4 activation to provide insights into the regulation of CO2-dependent breathing and to enable the development of small molecules that activate GPR4. In Aim 1, I will determine the molecular details of how protons activate GPR4. In Aim 2, I will decipher how current antagonists bind and inactive GPR4. To accomplish these aims, I will combine the techniques of biochemistry, structural biology (cryogenic electron microscopy), and pharmacology (mutagenesis and signaling assays). These studies will provide mechanistic insight into the regulation of GPR4 activity through protons and small molecules. This will expand our fundamental understanding of how protons drive CO2-dependent breathing, and this will enable the future development of small molecules that activate GPR4, which could be novel therapeutics for CCHS and CSA.