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.

抽象的 A-激酶锚定蛋白 (AKAP) 组织第二信使响应酶来引导 这些蛋白质最初被发现是作为蛋白激酶 A (PKA) 结合伴侣。 然而，现在很清楚，AKAP 的主要功能是整合各种细胞内信号。 通过将 PKA 与其他激酶、小 GTP 酶和蛋白磷酸酶隔离在以下范围内而发生 它们的底物。该机制的生理意义已在多种情况下得到验证。 该提案利用有关 AKAP220 信号复合物的新发现来确定是否操纵 这种大分子组装体在恢复水稳态方面具有治疗价值 管理常染色体显性多囊肾病的各个方面。 人体肾脏每天过滤约 180 升液体，但大部分水被重新吸收，因为只有 1.5 升 升尿液被排出体外，尿液被浓缩在肾脏收集管中，水被重新吸收。 通过水通道蛋白 2 (AQP2) 水孔的腔内液体 精氨酸加压素 (AVP) 增加。 通过诱导 AQP2 上 Ser256 的 PKA 磷酸化来刺激水的易位，从而实现水渗透性 从囊泡到集合管顶膜的孔，aquaporin-2 的缺陷并不奇怪。 贩运具有可怕的病理生理后果。 当充满液体的囊肿在肾脏集合管中生长时，就会发生多囊肾病。 最终取代大部分肾脏并导致常染色体显性多囊肾衰竭。 在分子水平上，纤毛突变是导致全球终末期肾衰竭的主要原因。 跨膜蛋白多囊蛋白 1 (PC1) 和多囊蛋白 2 (PC2) 通过促进渗透调节缺陷 aquaporin-2 异常的 cAMP 信号传导、改变纤毛组装并进一步降低 Rho GTP 酶活性 我们的初步发现表明 AKAP220 结合。 这些病理反应中的每一种的伙伴。 提出了三个具体目标的实验计划，目标 1 将采用最先进的分析和方法。 邻近连接方法来定义酶的组成、化学计量和亚细胞位置 AKAP220 复合物将研究 3D 类器官培养物以确定 AKAP220 是否与 GTP 酶相关。 效应蛋白 IQGAP1 通过 RhoA 的局部调节来维持肌动蛋白屏障的形成。 在微流体“芯片肾”装置中将药物递送至经过 CRISPR/Cas 9 基因编辑的肾源性细胞， 确定 AKAP220 相关磷酸酶 1 (PP1) 是否影响水通道蛋白 2 背后的信号转导事件 贩运和纤毛生物发生。