Gpr116 Regulation of Renal Acid Excretion

项目摘要 G蛋白偶联受体（GPCR）是一个不同的家族的完整的膜蛋白，识别一个 细胞外分子的分类，包括神经递质，激素，光和气味。故该等股份认购 GPCR是重要的药物靶点，有超过1200种FDA批准的药物靶向GPCR。但不少 GPCR具有未知的生物学作用。因此，揭示的功能和生理意义， 未充分研究的GPCR代表了大量未开发的治疗潜力。我们的实验室先前报告说， 粘附类GPCR（aGPCR），Gpr116，是肾中最高表达的GPCR之一。 然而，其在肾脏生理学中的作用尚未研究。最近发表的数据显示， 目前的建议表明，Gpr116定位于A-嵌入细胞（AIC）的顶膜， 鼠集合管此外，使用靶向Cre-Lox重组从AIC中缺失Gpr116， 系统显示Gpr116是肾脏酸排泄的关键调节剂。Gpr116缺陷小鼠 肾脏具有显著降低尿pH，且在诱导一种 代谢性酸中毒，表明Gpr116的丢失足以最大限度地酸化尿液。特别是 尿液pH值的降低伴随着血液pH值的升高和pCO2（酸/碱）的降低。 我们称之为"肾小管增生"。此外，Gpr116-null动物具有显著更多的表面 AIC中V-ATP酶质子泵的表达。尽管这些发现意义重大，但仍有 关于Gpr116在肾脏中的功能，我们的知识存在实质性空白。例如分子 在AIC中缺乏Gpr116的情况下导致尿液酸化的机制尚不清楚， 受体的内源性激活剂还有待鉴定。因此，本提案的总体目标 是为了解决这些关键组成部分的Gpr116肾生理。根据以前的观察和 在这个建议中概述了强有力的初步数据，我假设Gpr116激活是由以下因素促进的： 刺激AIC微绒毛中的膜结合糖蛋白，导致Rho GT3级联反应， 从腔膜回收V-ATP酶。这些支持了我的中心假设，即Gpr116作为一种 负反馈元件，通过调节V-ATP酶的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设：1）确定Gpr116激活的途径 抑制V-ATP酶表面表达;和2）鉴定促进V-ATP酶表面表达的相互作用和管腔条件。 AIC中Gpr116的内源性激活。这些研究的完成将确定分子途径 与肾脏中的Gpr116相关，并建立Gpr116作为一种治疗药物的治疗潜力。 肾脏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.