D1 and AT1 Receptor Interaction--Hypertension--mechanism

Felder项目的总体目标是研究在血压调节中起关键作用的两种肾钠调节途径之间的相互作用。这些通路的缺陷与高血压的发展直接相关。肾多巴胺能和血管紧张素系统独立地和一致地调节大部分肾钠排泄。我们将确定多巴胺D1和血管紧张素AT 1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钠转运的机制。天然存在的单核苷酸多态性G448 T、C679 T和C1711 T分别导致G蛋白偶联受体激酶4 γ同种型（GRK 4），特别是γ同种型中的氨基酸取代R65 L、A142 V和A486 V，组成性地增加酶活性，导致人肾中D1多巴胺受体从其G蛋白和效应物复合物磷酸化和解偶联。这些GRK 4基因变异体本身或通过其与肾素-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。Felder项目的总体假设是GRK 4 γ中的这些氨基酸变化组成性地使D1受体而不是AT 1受体脱敏，从而允许不受约束的AT 1受体作用。三个具体的目标已经开发，以确定GRK 4 γ变体对D1和AT 1受体在人近端小管细胞的差异作用的机制。1.我们将确定GRK 4 γ活性增加的机制 携带氨基酸取代R65 L、A142 V和A486 V。这将检验GRK 4 γ变体与D1受体的结合增加和/或GRK 4变体对质膜的靶向增加会增加GRK 4活性的假设。2.我们还将确定GRK 4 γ或其变体（R65 L，A142 V和A486 V）是否调节AT 1受体。这将检验GRK 4 γ变体不会使AT 1受体脱敏并允许AT 1受体在高血压中不受阻碍的作用的假设。3.我们将研究D1和AT 1受体的急性和慢性反式调节的基本机制。这将检验D1受体负调节AT 1受体的功能和表达以及这种调节在遗传性高血压中受损的假设。研究将在细胞中进行 内源地（例如，来自血压正常和高血压人的肾近端小管细胞，并与啮齿动物进行比较），以及异源表达D1和AT 1受体和GRK 4 γ变体的细胞，使用药理学、细胞和分子生物学工具和终点来研究D1和AT 1受体功能。基于我们对GRK 4 γ变体在D1受体信号传导中的重要作用的研究，我们认为，一个单一的基因，调节许多其他基因可能是原发性高血压的发病机制的主要贡献者。

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.