AKAP Modulation of Renal Signaling

摘要 A-激酶锚定蛋白(AKAP)组织第二信使反应酶来引导 单元格内的信息。这些蛋白最初被发现为蛋白激酶A(PKA)结合伙伴。 然而，现在已经清楚的是，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酶和蛋白磷酸酶隔离在以下范围内发生 它们的底物。这一机制的生理意义已经在几种情况下得到了验证。这 Proposal利用关于AKAP220信号复合体的新发现来确定是否操纵 这种大分子组装在恢复水的动态平衡方面具有治疗价值 常染色体显性遗传性多囊肾病的治疗。 人类肾脏每天过滤大约180升液体，但大部分水被重新吸收，因为只有1.5升 公升的尿液会被排出体外。尿液集中在肾脏收集管中，从那里重新吸收水分。 腔内液体通过水通道蛋白-2(AQP2)水孔。精氨酸加压素(AVP)激素增加 通过诱导AQP2上的PKA磷酸化Ser256刺激水的移位来实现水的通透性 从小泡到集合管顶膜的小孔。毫不奇怪，水通道蛋白-2的缺陷 人口贩运具有可怕的病理生理后果。 当充满液体的囊泡在肾脏收集管中生长时，就会发生多囊肾病。这些囊肿 最终替换大部分肾脏并导致肾衰竭。常染色体显性遗传性多囊肾 疾病是全世界终末期肾功能衰竭的主要原因。在分子水平上，纤毛的突变 跨膜蛋白多囊蛋白1(PC1)和多囊蛋白2(PC2)通过 水通道蛋白-2。CAMP信号异常，纤毛组装改变，Rho GTP酶活性进一步降低 有助于扩张充满液体的包囊。我们的初步发现涉及AKAP220结合 这些病理性反应中的每一个都是伙伴。 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 近距离连接方法确定酶的组成、化学计量和亚细胞定位 AKAP220复合体。AIM 2将研究3D有机物培养以确定AKAP220相关的GTP酶 效应蛋白IQGAP1通过局部调节RhoA来支持肌动蛋白屏障的形成。AIM 3将聘用 在微流控“芯片上肾”装置中将药物输送到CRISPR/Cas 9基因编辑的肾脏来源细胞中 确定AKAP220相关的磷酸酶1(PP1)是否影响水通道蛋白-2背后的信号事件 贩运和纤毛生物发生。

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.