Gpr116 Regulation of Renal Acid Excretion

项目摘要 G蛋白偶联受体(GPCRs)是一个不同的完整的膜蛋白家族，识别一个 各种细胞外分子，包括神经递质、激素、光线和气味。因此，他们 是重要的药物靶点，FDA批准的1200多种药物针对GPCRs。然而，许多人 GPCRs具有未知的生物学作用。由此，揭示了肌动蛋白的功能和生理意义 未得到充分研究的GPCRs代表了大量尚未开发的治疗潜力。我们的实验室此前报告说 粘附型gpcr(AGPCR)Gpr116是肾脏中表达最高的gpcr之一。 然而，它在肾脏生理学中的作用还没有被研究。最近发布的数据显示在 目前的建议证明Gpr116定位于A细胞的顶膜。 小鼠的收集管道。此外，使用靶向Cre-Lox重组从AICS中删除Gpr116 系统显示GPR116是肾酸排泄的关键调节因子。Gpr116基因缺陷的小鼠 肾脏的尿液pH值显著降低，并且在诱导AFP后不能进一步酸化他们的尿液 代谢性酸中毒，提示GPR116的丢失足以最大限度地酸化尿液。值得注意的是， 尿液酸碱度的降低伴随着血液酸碱度的升高和二氧化碳分压的降低。 我们称之为“肾小管性碱中毒”。此外，Gpr116缺失的动物有明显更多的表面积 V-ATPase质子泵在AICS中的表达。尽管这些发现意义重大，但仍有 我们对Gpr116在肾脏中的功能的认识存在很大差距。例如，分子 在缺乏Gpr116的AICS中导致尿酸的机制尚不清楚，而且 该受体的内源性激活剂尚未确定。因此，这项提案的首要目标是 是为了解决Gpr116肾脏生理学的这些关键组成部分。基于之前的观察和 在这项提案中概述的强大的初步数据，我假设Gpr116的激活是通过 刺激的AICS微绒毛中的膜结合糖蛋白，导致Rho GTP酶级联和 腔内膜V-ATPase的回收。这些都支持我的核心假设，即Gpr116作为一种 负反馈元件通过调节V-ATPase内吞作用来抑制过量的酸分泌。我 我提出了以下具体目标来验证这一假说：1)确定Gpr116激活的通路 抑制V-ATPase表面表达；以及2)识别相互作用和促进 AICS中Gpr116的内源性激活。这些研究的完成将确定分子途径 首次在肾脏中与Gpr116相关，并建立了Gpr116作为一种 肾酸排泄的靶向调节剂。

Project Summary G protein-coupled receptors (GPCRs) are a diverse family of integral membrane proteins that recognize an assortment of extracellular molecules including neurotransmitters, hormones, light and odors. Accordingly, they are important pharmaceutical targets, with over 1200 FDA approved drugs targeting GPCRs. However, many GPCRs have unknown biologic roles. Thus, uncovering the function and physiologic significance of understudied GPCRs represents a wealth of untapped therapeutic potential. Our lab previously reported that an adhesion-class GPCR (aGPCR), Gpr116, is among the most highly expressed GPCRs in the kidney. However, its role in renal physiology had not been investigated. Recently published data presented in the current proposal demonstrate localization of Gpr116 to the apical membrane of A-intercalated cells (AICs) in murine collecting ducts. Furthermore, deletion of Gpr116 from AICs using a targeted Cre-Lox recombination system revealed Gpr116 to be a critical regulator of renal acid excretion. Mice deficient for Gpr116 in the kidney have significantly reduced urine pH and are incapable of further acidifying their urine after induction of a metabolic acidosis, suggesting that loss of Gpr116 is sufficient to maximally acidify urine. Notably, the reduction in urine pH is accompanied with an increase in blood pH and a decrease in pCO2, an acid/base disorder we term “renal tubular alkalosis”. Moreover, Gpr116-null animals have significantly more surface expression of V-ATPase proton pumps in AICs. Although these findings are significant, there are still substantial gaps in our knowledge regarding the function of Gpr116 in the kidney. For example, the molecular mechanisms that cause urine acidification in the absence of Gpr116 in AICs are unknown, and the endogenous activator of the receptor has yet to be identified. Therefore, the overarching goal of this proposal is to address these critical components of Gpr116 renal physiology. Based on previous observations and strong preliminary data outlined in this proposal, I hypothesize that Gpr116 activation is facilitated by membrane-bound glycoproteins in the microvilli of stimulated AICs, leading to a Rho GTPase cascade and a retrieval of V-ATPase from the luminal membrane. These support my central hypothesis that Gpr116 acts as a negative-feedback element to inhibit excessive acid secretion through regulation of V-ATPase endocytosis. I have proposed the following specific aims to test this hypothesis: 1) Determine the Gpr116-activated pathways that inhibit V-ATPase surface expression; and 2) Identify interactions and luminal conditions that facilitate endogenous activation of Gpr116 in AICs. Completion of these studies will define the molecular pathways relevant to Gpr116 in the kidney for the first time and establish the therapeutic potential of Gpr116 as a targeted modulator of renal acid excretion.