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（AKAPS）组织了第二个使者反应式酶，以指导流动 细胞内的信息。这些蛋白质最初被发现为蛋白激酶A（PKA）结合伴侣。 但是，现在很明显，AKAP的主要功能是整合各种细胞内信号。这 通过与其他激酶，小的GTPases和蛋白质磷酸酶隔离PKA。 他们的底物。这种机制的物理意义已在多种情况下得到了验证。 提案利用有关AKAP220信号复合物的新发现，以确定是否操纵 这种大分子组件在恢复水稳态至 管理常染色体显性多囊性肾脏疾病的各个方面。 人肾脏每天过滤约180升液体，但大多数水被重吸收，仅为1.5 尿液是独家的。尿液集中在肾脏收集的管道中 通过水通道蛋白-2（AQP2）水孔的腔液流体。辣椒精氨酸加压素（AVP）增加 Ser256在AQP2上诱导的PKA磷酸化的水渗透性刺激水的易位 从蔬菜到收集管道的根尖膜的毛孔。毫不奇怪，Aquaporin-2中的缺陷 贩运有严重的病理生理后果。 当在肾脏收集管道中生长液体囊肿时，会发生多囊肾脏疾病。这些囊肿 最终取代了许多孩子，并导致肾脏衰竭。常染色体显性多囊肾脏 疾病是全球终末期肾衰竭的主要原因。在分子水平上，纤毛中的突变 跨膜蛋白多囊蛋白1（PC1）和多囊蛋白2（PC2）通过通过 Aquaporin-2。异常的营地信号传导，改变纤毛组装和进一步的Rho GTPase活性降低 有助于填充囊肿的扩展。我们的初步发现植入AKAP220结合 这些病理反应中的每一个。 提出了三个特定目标的实验计划。 AIM 1将采用最先进的分析和 定义酶成分，化学计量和亚细胞位置的接近连接方法 AKAP220复合物。 AIM 2将研究3D器官培养物，以确定AKAP220相关的GTPase是否存在 效应蛋白IQGAP1通过RhoA的局部调节自杀肌动蛋白屏障形成。 AIM 3将使用 在微流体的“肾脏芯片”设备中，将药物输送到CRISPR/CAS 9基因编辑的肾脏衍生细胞到 确定AKAP220相关的磷酸酶1（PP1）是否会影响基于Aquaporin-2的信号事件 贩运和纤毛生物发生。