AKAP Modulation of Renal Signaling

肾脏信号传导的 AKAP 调节

• 批准号: 9816376
• 负责人: John D Scott
• 金额: $ 34.09万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9816376
• 关键词: 3-Dimensional; A kinase anchoring protein; Actins; Argipressin; Autosomal Dominant Polycystic Kidney; Binding; Biological Assay; Blood Plasma Volume; CRISPR/Cas technology; Calcium; Cell Line; Cells; Cilia; Complex; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyst; Defect; Development; Devices; Drug Delivery Systems; Duct (organ) structure; Electrolyte Balance; End stage renal failure; Engineering; Enzymes; Equilibrium; Event; Genes; Guanosine Triphosphate Phosphohydrolases; HDAC1 gene; HDAC6 gene; Holoenzymes; Homeostasis; Hormones; Human; Hydration status; IQ motif containing GTPase activating protein 1; In Vitro; Individual; Integral Membrane Protein; Kidney; Kidney Failure; Lead; Ligation; Liquid substance; Location; Membrane; Microfluidics; Molecular; Molecular Profiling; Monomeric GTP-Binding Proteins; Morphogenesis; Movement; Mus; Mutation; Organ; Organoids; Osmoregulation; PKD2 protein; Pathologic; Pathology; Patients; Permeability; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Photobleaching; Physiological; Polycystic Kidney Diseases; Protein Dephosphorylation; Protein phosphatase; Proteins; Renal Tissue; Renal function; Second Messenger Systems; Signal Transduction; Site; Stem cells; Testing; Therapeutic; Tissue Engineering; Urine; Vasopressins; Vesicle; Water; apical membrane; aquaporin-2; cellular engineering; cilium biogenesis; collecting tubule structure; experience; glomerular filtration; kidney cell; macromolecular assembly; polycystic kidney disease 1 protein; preservation; prospective; response; restoration; rho; rho GTP-Binding Proteins; single molecule; stoichiometry; trafficking

ABSTRACT A-Kinase Anchoring Proteins (AKAPs) organize second messenger responsive enzymes to direct the flow of information within cells. These proteins were initially discovered as protein kinase A (PKA) binding partners. However, it is now clear that the primary function of AKAPs is to integrate a variety of intracellular signals. This occurs by sequestering PKA with other kinases, small GTPases, and protein phosphatases within range of their substrates. The physiological significance of this mechanism has been validated in several contexts. This proposal exploits new discoveries about AKAP220 signaling complexes to establish if manipulation of this macromolecular assembly is of therapeutic value in the restoration of water homeostasis to manage aspects of autosomal dominant polycystic kidney disease. Human kidneys filter about 180 liters of fluid every day, yet a majority of the water is reabsorbed, as only 1.5 liters of urine is excreted. Urine is concentrated in the kidney-collecting duct where water is reabsorbed from luminal fluid through aquaporin-2 (AQP2) water pores. The hormone arginine vasopressin (AVP) increases water permeability by inducing PKA phosphorylation of Ser256 on AQP2 to stimulate translocation of the water pore from vesicles to apical membranes of collecting ducts. Not surprisingly, defects in aquaporin-2 trafficking have dire pathophysiological consequences. Polycystic kidney disease occurs when fluid filled cysts grow in the kidney collecting ducts. These cysts eventually replace much of the kidneys and lead to kidney failure. Autosomal dominant polycystic kidney disease is a leading cause of end-stage renal failure worldwide. At the molecular level, mutations in the cilia transmembrane proteins polycystin 1 (PC1) and polycystin 2 (PC2) promote defective osmoregulation through aquaporin-2. Aberrant cAMP signaling, altered cilia assembly and reduced Rho GTPase activity further contribute to the expansion of fluid filled cysts. Our preliminary findings implicate AKAP220-binding partners in each of these pathological responses. An experimental plan of three specific aims is proposed. Aim 1 will employ state-of-the-art analytical and proximity ligation approaches to define the enzyme composition, stoichiometry and subcellular location of AKAP220 complexes. Aim 2 will investigate 3D organoid cultures to establish if AKAP220-associated GTPase effector protein IQGAP1 sustains actin barrier formation through local modulation of RhoA. Aim 3 will employ drug delivery to CRISPR/Cas 9 gene-edited kidney-derived cells in a microfluidic “Kidney-on-a-chip” device to determine if AKAP220-associated phosphatase 1 (PP1) impacts signaling events that underlie aquaporin-2 trafficking and cilia biogenesis.

摘要 A-激酶介导蛋白（AKAP）组织第二信使应答酶，以指导细胞内的细胞流动。 细胞内的信息。这些蛋白最初被发现作为蛋白激酶A（PKA）结合伴侣。 然而，现在清楚的是，AKAP的主要功能是整合各种细胞内信号。这 通过将PKA与其他激酶、小GTP酶和蛋白磷酸酶在 它们的基质。这种机制的生理意义已经在几种情况下得到验证。这 该提案利用了关于AKAP 220信号复合物的新发现， 这种大分子组装体在恢复水体内平衡方面具有治疗价值， 多囊肾的病因有哪些？ 人类肾脏每天过滤约180升液体，但大部分水被重吸收，因为只有1.5升被重吸收。 排泄的尿液为100升。尿液集中在肾集合管中，在那里水被重新吸收， 腔液通过水通道蛋白-2（AQP 2）的水孔。精氨酸加压素（AVP）增加 通过诱导AQP 2上Ser 256的PKA磷酸化来刺激水的转运， 从小泡到集合管顶端膜的孔。毫不奇怪，水通道蛋白-2的缺陷 贩运人口具有可怕的病理生理后果。 多囊肾是一种常见的肾脏疾病。这些囊肿 最终取代大部分肾脏并导致肾衰竭。常染色体显性多囊肾 疾病是全世界终末期肾衰竭的主要原因。在分子水平上，纤毛的突变 跨膜蛋白多囊蛋白1（PC 1）和多囊蛋白2（PC 2）通过以下途径促进有缺陷的β-淀粉样蛋白调节： 水通道蛋白-2。异常的cAMP信号传导，改变的纤毛组装和降低的Rho GT3活性进一步 导致充满液体的囊肿扩张。我们的初步发现暗示AKAP 220结合 这些病理反应中的每一个。 提出了三个具体目标的实验方案。目标1将采用最先进的分析和 邻位连接方法来确定酶的组成、化学计量和亚细胞定位， AKAP 220复合物。目标2将研究3D类器官培养物，以确定AKAP 220是否与GTPase相关 效应蛋白IQGAP 1通过局部调节RhoA维持肌动蛋白屏障的形成。目标3将采用 药物递送到微流体“芯片上的肾脏”装置中的CRISPR/Cas 9基因编辑的肾脏衍生细胞， 确定AKAP 220相关磷酸酶1（PP 1）是否影响水通道蛋白-2基础的信号传导事件 运输和纤毛生物发生。