D1 and AT1 Receptor Interaction--Hypertension--mechanism

该项目的总体目标是研究在血压调节中起关键作用的两条肾脏钠调节通路之间的相互作用。这些通路的缺陷与高血压的发展直接相关。肾脏的多巴胺能系统和血管紧张素系统独立地和协同地调节大部分肾脏的钠排泄。我们将确定多巴胺D1和血管紧张素AT1受体的相互作用通过共同和独立的第二信使途径调节血压和肾脏钠转运的机制。自然发生的单核苷酸多态G448T、C679T和C1711T导致G蛋白偶联受体激酶4伽马亚型(GRK4)的氨基酸替换R65L、A142V和A486V，特别是伽马亚型，结构性地增加酶活性，导致人类肾脏中D1多巴胺受体与其G蛋白和效应复合体的磷酸化和解偶联。这些GRK4基因变异本身或通过它们与肾素-血管紧张素系统的相互作用，在人类(和啮齿动物)遗传性高血压的发病机制中起着至关重要的作用。费尔德项目的总体假说是，GRK4Gamma中的这些氨基酸变化构成了对D1受体的失敏，而不是AT1受体，从而允许AT1受体不受限制地发挥作用。为了确定GRK4变异体对人近端肾小管上皮细胞D1和AT1受体的不同作用机制，已经开发了三个特定的靶点。1.我们将确定GRK4Gamma活性升高的机制 携带氨基酸替代，R65L，A142V和A486V。这将检验这样一种假设，即增加GRK4变异体与D1受体的结合和/或增加GRK4变异体对质膜的靶向性会增加GRK4的活性。2.我们还将确定GRK4Gamma或其变体(R65L、A142V和A486V)是否调节AT1受体。这将检验GRK4伽马变种不会使AT1受体脱敏并允许AT1受体在高血压中不受阻碍地发挥作用的假设。3.研究D1和AT1受体急、慢性反式调节的基本机制。这将检验一种假设，即D1受体负向调节AT1受体的功能和表达，而这种调节在遗传性高血压中受到损害。研究将在细胞中进行 利用药理学、细胞和分子生物学工具和终点，在内源性(例如，正常血压和高血压人的肾近端小管细胞，并与啮齿动物比较)以及异源表达D1和AT1受体和GRK4伽马变异体的细胞中，研究D1和AT1受体的功能。基于我们对GRK4变异体在D1受体信号转导中的重要作用的研究，我们认为，调节许多其他基因的单个基因可能是原发性高血压发病的主要因素。

The overall goal of Project by Felder is to study the interaction between two renal sodium regulatory pathways that play a key role in blood pressure regulation. Defects in these pathways have been directly linked to the development of hypertension. The renal dopaminergic and angiotensin systems independently and in concert regulate most of renal sodium excretion. We will determine the mechanisms by which the interactions of dopamine D1, and angiotensin AT1 receptors regulate blood pressure and renal sodium transport through common and independent second messenger pathways. Naturally occurring single nucleotide polymorphisms, G448T, C679T, and C1711T that result in amino acid substitutions R65L, A142V, and A486V, respectively in G protein-coupled receptor kinase 4 gamma isoform (GRK4), specifically of the gamma isoform, constitutively increase enzyme activity, resulting in the phosphorylation and uncoupling of the D1 dopamine receptor from its G protein and effector complexes in the human kidney. These GRK4 gene variants, by themselves or via their interactions with the renin-angiotensin system, play a crucial role in the pathogenesis of human (and rodent) genetic hypertension. The over all hypothesis of Project by Felder is that these amino acid changes in GRK4gamma constitutively desensitize D1 receptors but not AT1 receptors, allowing unfettered AT1 receptor action. Three specific aims have been developed to determine the mechanisms of the differential actions of GRK4gamma variants on D1 and AT1 receptors in human proximal tubule cells. 1. We will determine the mechanism of the increased activity of GRK4gamma carrying amino acid substitutions, R65L, A142V, and A486V. This will test the hypothesis that increased binding of GRK4gamma variants to D1 receptors and/or increased targeting of GRK4 variants to the plasma membrane increase GRK4 activity. 2. We will also determine whether GRK4gamma or its variants (R65L, A142V, and A486V) regulate AT1 receptors. This will test the hypothesis that GRK4gamma variants do not desensitize AT1 receptors and allow unimpeded AT1 receptor actions in hypertension. 3. We will study the fundamental mechanisms involved in the acute and chronic transregulation of D1 and AT1 receptors. This will test the hypothesis that D1 receptors negatively regulate the function and expression of AT1 receptors and that this regulation is impaired in genetic hypertension. Studies will be performed in cells endogenously (e.g., renal proximal tubule cells from normotensive and hypertensive humans and compared to rodents), as well as in cells heterologously expressing D1 and AT1 receptors and GRK4gamma variants using pharmacological, cellular and molecular biological tools and end points to study D1 and AT1 receptor function. Based on our studies on the important role of GRK4gamma variants in D1 receptor signaling, we suggest that a single gene that regulates many other genes may be a major contributor to the pathogenesis of essential hypertension.