H+-ATPase And AQP2: Regulation Of Targeting And Recycling In IMCD Cells

H-ATP酶和AQP2：IMCD细胞中靶向和回收的调节

• 批准号: 7885537
• 负责人: JOHN H SCHWARTZ
• 金额: $ 35.55万
• 依托单位: BOSTON MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7885537
• 关键词: Acid-Base Equilibrium; Acids; Adaptor Signaling Protein; Adenylate Cyclase; Affinity; Amino Acids; Apical; Binding; Binding Proteins; Biological Assay; C-terminal; Cell membrane; Cells; Chimera organism; Chimeric Proteins; Clathrin; Complex; Consensus; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Development; Disease; Dose; Endocytosis; Exocytosis; Forskolin; Genetic; Intervention Studies; Kidney; Lead; Length; Liquid substance; Mediating; Membrane; Membrane Transport Proteins; Modality; Molecular; Mutate; N-terminal; Phosphorylation; Process; Protein Binding; Protein Dephosphorylation; Protein Isoforms; Proteins; Proton-Translocating ATPases; Protons; Rattus; Recycling; Regulation; Rest; Retrieval; Role; S-nitro-N-acetylpenicillamine; SNAP receptor; SNAPIN gene; Signal Transduction; Site; System; Testing; Therapeutic Intervention; Transmembrane Transport; Transport Process; Vasopressins; Vesicle; Water; apical membrane; aquaporin-2; base; coated pit; expectation; human SNAP23 protein; improved; in vivo; inhibitor/antagonist; mutant; novel; overexpression; prevent; public health relevance; receptor; response; syntaxin; syntaxin 1A; target SNARE proteins; trafficking

DESCRIPTION (provided by applicant): IMCD cells in vivo and in culture secrete H+, mediated by an H+-ATPase, and absorb H2O mediated by aquaporin-2 (AQP2). In cultured rat IMCD cells and in intact IMCD segments, transport is regulated by SNARE mediated exocytic insertion and endocytic retrieval of vesicles carrying either the H+-ATPase or AQP2. Although exocytosis of H+-ATPase and AQP2 are independently regulated, they utilize a similar SNARE system for membrane targeting-fusion. In this proposal we will characterize the targeting process for these proteins and identify the disparate effects of SNARE regulators on H+-ATPase versus AQP2 membrane cycling. We will test the hypothesis that the B1 subunit of the plasma membrane H+-ATPase contains molecular information for targeting of the assembled H+-ATPase to the apical membrane. B1 is the isoform of the B subunit that is present in the H+-ATPase of cells specialized for proton secretion. The other isoform, B2, is present in the vesicular membrane H+-ATPase of all cells. We predict that the targeting information for the plasma membrane H+-ATPase is encoded in either the N or the C-terminal amino acids, the two regions of ATP6V1B1 that are most dissimilar from B2. Our studies will also determine the role of Munc 18-2 and snapin as SNARE regulators for H+- ATPase and AQP2. Munc 18-2, through PKC signaling, controls the apical targeting and insertion of H+-ATPase in IMCD cells by regulating its interaction with syntaxin. We hypothesize that Munc 18-2 controls plasma membrane AQP2 very differently by regulating both AQP2 exocytosis and endocytosis. We propose the following cycle: Munc prevents formation of a fusion complex by association with vesicular AQP2-VAMP. Upon PKA phosphorylation of AQP2, Munc dissociates from AQP2 and VAMP and vesicle fusion occurs delivering AQP2, VAMP and Munc to the plasma membrane. Upon dephosphorylation of plasma membrane AQP2, Munc reassociates with AQP2, concentrates AQP2 and VAMP in clathrin-coated pits where this complex of Munc-AQP2-VAMP is endocytosed into the cell interior. Lastly, the role of snapin, a recently identified SNAP and adenylate cyclase binding protein, will be evaluated. We propose that snapin regulates exocytosis of both H+-ATPase and AQP2 in IMCD cells. Upon snapin binding to SNAP-23, an increased number of SNAP-syntaxin complexes are formed which are the t-SNARE receptor for the vesicular VAMP, delivering H+-ATPase to the apical membrane. Snapin may also regulate AQP2 exocytosis by amplifying the vasopressin second message (cAMP) since it has been shown that upon PKA phosphorylation, snapin binds and activates adenylate cyclase. We believe these studies will provide novel paradigms for the regulation of these apical membrane transporters and provide newer modalities for the treatment of fluid and acid-base disorders. PUBLIC HEALTH RELEVANCE These studies will provide important new information concerning the mechanisms by which the kidney regulates water and acid-base balance and how these mechanisms are disrupted by genetic and acquired disease states. An enhanced understanding of these abnormalities could lead to the development of improved therapeutic interventions.

描述（由申请人提供）：体内和培养的IMCD细胞通过H+-ATP酶介导分泌H+，并通过水通道蛋白-2（AQP 2）介导吸收H2O。在培养的大鼠IMCD细胞和完整的IMCD节段中，运输受SNARE介导的携带H+-ATP酶或AQP 2的囊泡的胞吐插入和胞吞回收的调节。虽然H+-ATP酶和AQP 2的胞吐作用是独立调节的，但它们利用类似的SNARE系统进行膜靶向融合。在这项提案中，我们将表征这些蛋白质的靶向过程，并确定SNARE调节剂对H+-ATP酶与AQP 2膜循环的不同影响。我们将测试的假设，质膜H+-ATP酶的B1亚基包含的分子信息的目标组装的H+-ATP酶的顶膜。B1是B亚单位的同种型，存在于质子分泌专用细胞的H+-ATP酶中。另一种亚型B2存在于所有细胞的囊泡膜H+-ATP酶中。我们预测质膜H+-ATP酶的靶向信息编码在N或C末端氨基酸中，这两个区域与B2最不相似。我们的研究还将确定Munc 18-2和snapin作为H+-ATP酶和AQP 2的SNARE调节剂的作用。Munc 18-2通过PKC信号传导，通过调节H+-ATP酶与突触融合蛋白的相互作用来控制IMCD细胞中H+-ATP酶的顶端靶向和插入。我们推测Munc 18-2通过调节AQP 2的胞吐和胞吞作用来非常不同地控制质膜AQP 2。我们提出以下循环：Munc通过与囊泡AQP 2-VAMP结合来防止融合复合物的形成。在AQP 2的PKA磷酸化后，Munc从AQP 2和VAMP解离，并且发生囊泡融合，将AQP 2、VAMP和Munc递送至质膜。在质膜AQP 2去磷酸化后，Munc与AQP 2重新结合，将AQP 2和VAMP浓缩在网格蛋白包被的凹坑中，在该处Munc-AQP 2-VAMP的复合物被内吞到细胞内部。最后，snapin，最近确定的SNAP和腺苷酸环化酶结合蛋白的作用，将进行评估。我们建议，snapin调节IMCD细胞的H+-ATP酶和AQP 2的胞吐作用。在与SNAP-23结合后，形成数量增加的SNAP-突触融合蛋白复合物，其是囊泡VAMP的t-SNARE受体，将H+-ATP酶递送至顶膜。Snapin还可以通过放大加压素第二信使（cAMP）来调节AQP 2的胞吐作用，因为已经表明，在PKA磷酸化后，Snapin结合并激活腺苷酸环化酶。我们相信这些研究将为这些顶端膜转运蛋白的调节提供新的范例，并为治疗液体和酸碱失衡提供新的模式。 公共卫生相关性 这些研究将提供有关肾脏调节水和酸碱平衡的机制以及这些机制如何被遗传和获得性疾病状态破坏的重要新信息。对这些异常的进一步了解可能会导致改进治疗干预措施的发展。