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

• 批准号: 7792377
• 负责人: Sathyamangla V Prasad
• 金额: $ 38.63万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7792377
• 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

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(申请人提供)：β-肾上腺素能受体(BAR)信号是心功能最强大的调节因子之一。在人类心力衰竭中，质膜上受体数量的减少与G蛋白偶联(脱敏)受损相关，导致对神经激素的反应性降低。受体脱敏是由bar kinase1(BARK1)对激动剂激活的bar的磷酸化启动的。B-arrestin与磷酸化的受体结合，导致效应因子(腺苷环化酶)信号的丢失。BAR复合体的靶向是内吞作用，导致内体隔室的受体去磷酸化，然后再循环回到质膜。以前我们已经证明，bARK1与磷脂酰肌醇3-激酶(PI3K)相互作用，形成一个针对PI3K的胞浆复合体，靶向激活的受体，其中PI3K在受体内吞作用中发挥作用。我们现在已经在小鼠体内发现了一种新的受体再敏感现象，这种现象是通过心脏过度表达一个非活性的PI3K突变体来实现的。此外，我们已经证明，BAR再增敏是有益的，因为它通过保留BAR功能来防止有害的心脏重构。这些初步数据与目前受体再增敏的范式相反，该范式表明，磷酸化-脱敏的受体必须经过内化才能被去磷酸化，然后才能循环回到质膜。调控这种新的质膜受体再增敏现象的分子机制尚不清楚。我们假设受体靶向PI3K活性负向调节受体在质膜上的再敏化。因此，抑制受体定位的PI3K活性会导致受体在质膜上重新增敏，而不需要内化。本研究的具体目的如下：1)确定PI3K活性是否调节bar的再敏化。通过G蛋白偶联、受体磷酸化、腺苷环化酶活性/cAMP水平的受体再敏化的详细分析将使用含有蛋白质或/和脂激酶活性的PI3K突变体进行。2)探讨PI3K是否通过调节蛋白磷酸酶活性实现BAR的复敏作用。将对PI3K突变体和抑制剂(体内和体外)对蛋白磷酸酶活性(PP1、PP2A等)的调节进行分析，以确定PI3K活性的作用。3)探讨PI3K调节PP2A活性的分子机制。通过脂质结合研究、代谢标记、RNAi敲除以及PP2A和I2-PP2A突变体的使用，将深入研究PP2A亚单位和蛋白磷酸酶抑制物(I-PP2As)的脂质结合或磷酸化对磷酸酶活性的调节。项目简介：B-肾上腺素能受体(BARS)是细胞表面最大的受体家族，是心脏功能最强的调节剂之一。人类心力衰竭的特征是BAR的下调(细胞表面丢失)和慢性脱敏(受体无法发出信号)。虽然脱敏现象已经得到了很好的研究，但重新增敏(受体恢复活力并对信号产生能力的过程)还没有被很好地理解。我们拨款提案中的研究将勾勒出这一新机制。阐明这一机制将导致识别分子，使我们能够通过靶向再增敏来开发新的心力衰竭治疗策略。这一点至关重要，因为目前的大多数治疗策略都涉及以受体为靶点来减弱下调调控。重要的是，确定再增敏的机制将具有广泛的普遍性，因为它适用于其他G蛋白偶联受体。