Novel regulation of beta-adrenergic receptor function by phosphoinositide 3-kinas

磷酸肌醇 3-激酶对 β-肾上腺素能受体功能的新调节

基本信息

批准号：
7839075
负责人：
Sathyamangla V Prasad
金额：
$ 23.48万
依托单位：
CLEVELAND CLINIC LERNER COM-CWRU
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-15 至 2011-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7839075
关键词：
1-Phosphatidylinositol 4-Kinase Adenylate Cyclase Adrenergic Receptor Agonist Applications Grants Arrestins Attenuated Back Beta-Adrenergic Receptor Kinase 1 Binding Biological Preservation Cardiac Catecholamines Cell Surface Receptors Cell membrane Cell surface Chronic Complex Coupling Cyclic AMP Data Down-Regulation Endocytosis Family G-Protein-Coupled Receptors GTP-Binding Proteins Heart failure Hormones Human In Vitro Label Lead Lipid Binding Lipids Mediating Molecular Mus Phosphatidylinositols Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Plasma Play Process Protein Dephosphorylation Protein Phosphatase 2A Regulatory Subunit PR53 Protein Phosphatase Inhibitor Protein phosphatase Proteins RNA Interference Receptor Inhibition Receptor Signaling Recycling Regulation Research Role STK6 gene Signal Transduction Stress Therapeutic beta-adrenergic receptor desensitization in vivo inhibitor/antagonist knock-down metabolic abnormality assessment mutant novel novel therapeutics overexpression phosphatidylinositol receptor prevent receptor receptor downregulation receptor function

项目摘要

DESCRIPTION (provided by applicant): Beta-adrenergic receptor (bAR) signaling is one of the most powerful regulators of cardiac function. In human heart failure, diminished receptor numbers at the plasma membrane associated with impaired G-protein coupling (desensitization) results in reduced responsiveness to neuro-hormones. Receptor desensitization is initiated by the phosphorylation of agonist activated bARs by bAR kinase-1 (bARK1). b-arrestin binds to the phosphorylated receptor resulting in the loss of effector (adenylyl cyclase) signaling. The bAR complex is targeted for endocytosis resulting in dephosphorylation of the receptor in the endosomal compartment before being recycled back to the plasma membrane. Previously we have shown that bARK1 interacts with phosphoinositide 3-kinase (PI3K) to form a cytosolic complex targeting PI3K to the activated receptor where PI3K plays a role in receptor endocytosis. We have now uncovered a novel phenomenon of receptor resensitization at the plasma membrane in vivo in mice by cardiac overexpression of an inactive PI3K mutant. Furthermore, we have demonstrated that bAR resensitization is beneficial as it prevents deleterious cardiac remodeling through preservation of bAR function. These preliminary data are contrary to the current paradigm of receptor resensitization which articulates that phosphorylated-desensitized receptor has to undergo internalization to be dephosphorylated before being recycled back to the plasma membrane. The molecular mechanism regulating this novel phenomenon of plasma membrane receptor-resensitization is not known. We hypothesize that receptor targeted PI3K activity negatively regulates receptor resensitization at the plasma membrane. Therefore, inhibition of receptor localized PI3K activity results in receptor resensitization at the plasma membrane without the need for internalization. The following specific aims are proposed in this study: 1) To determine whether PI3K activity regulates bAR resensitization. Detailed analysis of receptor resensitization by G-protein coupling, receptor phosphorylation, adenylyl cyclase activity/cAMP levels will be performed using PI3K mutants containing protein or/and lipid kinase activity. 2) To delineate whether PI3K-mediated bAR resensitization occurs through regulation of protein phosphatase activity. Analysis on regulation of protein phosphatase activity (PP1, PP2A etc.,) (in vivo and in vitro) by PI3K mutants and inhibitors will be carried out to define the role of PI3K activity. 3) To determine the molecular mechanism underlying the regulation of PP2A activity by PI3K. In-depth studies on regulation of phosphatase activity by lipid binding or phosphorylation of PP2A subunits and inhibitors of protein phosphatases (I-PP2As) will be performed using lipid binding studies, metabolic labeling, RNAi knock down and the use of PP2A and I2-PP2a mutants. PROJECT NARRATIVE: b-adrenergic receptors (bARs) belong to the largest family of cell surface receptors and are one of the strongest regulators of cardiac function. Human heart failure is characterized by downregulation (loss from the cell surface) and chronic desensitization (inability of the receptor to signal) of bARs. While the phenomenon of desensitization is well studied, resensitization (a process by which receptors rejuvenate and become competent to signal) is not well understood. Studies in our grant proposal will delineate this novel mechanism. Elucidation of this mechanism will lead to identification of molecules that would allow us to develop novel therapeutic strategies for heart failure by targeting resensitization. This is critical because the majority of the current therapeutic strategies involve targeting the receptor to attenuate downregulation. Importantly, determining the mechanism of resensitization will have broad universality as it is applicable to other G-protein coupled receptors.

描述（由申请人提供）：β-肾上腺素能受体（bAR）信号传导是心脏功能最强大的调节剂之一。在人类心力衰竭中，与 G 蛋白偶联受损（脱敏）相关的质膜受体数量减少，导致对神经激素的反应性降低。受体脱敏是通过 bAR 激酶-1 (bARK1) 磷酸化激动剂激活的 bAR 来启动的。 b-arrestin 与磷酸化受体结合，导致效应器（腺苷酸环化酶）信号传导丧失。 bAR 复合物被靶向内吞作用，导致内体区室中的受体去磷酸化，然后再循环回质膜。之前我们已经证明 bARK1 与磷酸肌醇 3 激酶 (PI3K) 相互作用，形成一种胞质复合物，将 PI3K 靶向激活的受体，其中 PI3K 在受体内吞作用中发挥作用。我们现在发现了一种新的现象，即通过非活性 PI3K 突变体的心脏过度表达，使小鼠体内质膜受体重新敏化。此外，我们已经证明 bAR 再敏化是有益的，因为它可以通过保留 bAR 功能来防止有害的心脏重塑。这些初步数据与当前受体再敏化的范例相反，该范例阐明磷酸化脱敏受体在再循环回质膜之前必须经历内化以去磷酸化。调节这种质膜受体再敏化新现象的分子机制尚不清楚。我们假设受体靶向 PI3K 活性负向调节质膜上的受体再敏化。因此，抑制受体局部 PI3K 活性会导致质膜上的受体重新敏化，而不需要内化。本研究提出以下具体目标： 1) 确定 PI3K 活性是否调节 bAR 重新敏化。将使用含有蛋白质或/和脂质激酶活性的PI3K突变体对G蛋白偶联、受体磷酸化、腺苷酸环化酶活性/cAMP水平引起的受体再敏化进行详细分析。 2) 描述PI3K介导的bAR再敏化是否通过调节蛋白磷酸酶活性而发生。将进行 PI3K 突变体和抑制剂对蛋白磷酸酶活性（PP1、PP2A 等）（体内和体外）调节的分析，以确定 PI3K 活性的作用。 3) 确定PI3K调节PP2A活性的分子机制。将利用脂质结合研究、代谢标记、RNAi 敲除以及 PP2A 和 I2-PP2a 的使用，深入研究通过脂质结合或 PP2A 亚基磷酸化以及蛋白磷酸酶 (I-PP2As) 抑制剂来调节磷酸酶活性突变体。项目叙述：b-肾上腺素能受体 (bAR) 属于最大的细胞表面受体家族，是心脏功能最强的调节剂之一。人类心力衰竭的特点是 bAR 下调（从细胞表面丢失）和慢性脱敏（受体无法发出信号）。虽然脱敏现象已得到充分研究，但重新敏化（受体恢复活力并变得有能力发出信号的过程）尚不清楚。我们的拨款提案中的研究将描述这种新颖的机制。阐明这种机制将导致分子的鉴定，使我们能够通过靶向再敏化来开发新的心力衰竭治疗策略。这是至关重要的，因为当前的大多数治疗策略都涉及靶向受体以减弱下调。重要的是，确定再敏化机制将具有广泛的普适性，因为它适用于其他 G 蛋白偶联受体。