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: 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
• 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

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）信号是心功能最强大的调节因子之一。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。受体脱敏是由激动剂激活的bAR激酶-1 （bARK1）磷酸化引起的。b-阻滞蛋白与磷酸化受体结合，导致效应（腺苷酸环化酶）信号的丧失。bAR复合体的目标是内吞作用，导致内体腔室中受体的去磷酸化，然后再循环回质膜。先前我们已经证明bARK1与磷酸肌肽3-激酶（PI3K）相互作用，形成一个将PI3K靶向激活受体的胞质复合物，其中PI3K在受体内吞作用中起作用。我们现在已经发现了一种新的现象，即在小鼠体内通过心脏过表达无活性的PI3K突变体在质膜上的受体再敏化。此外，我们已经证明，bAR再敏化是有益的，因为它通过保留bAR功能来防止有害的心脏重塑。这些初步数据与目前的受体再敏化范式相反，该范式阐明了磷酸化脱敏受体必须经过内化才能在再循环回质膜之前去磷酸化。调控这一新的质膜受体再敏化现象的分子机制尚不清楚。我们假设受体靶向PI3K活性负调控受体在质膜上的再敏化。因此，抑制受体定位的PI3K活性会导致质膜上的受体再敏化，而不需要内化。本研究提出以下具体目的：1)确定PI3K活性是否调节bAR再敏化。通过g蛋白偶联、受体磷酸化、腺苷酸环化酶活性/cAMP水平进行受体再敏化的详细分析将使用含有蛋白质或/和脂质激酶活性的PI3K突变体。2)阐明pi3k介导的bAR重敏是否通过调节蛋白磷酸酶活性发生。通过分析PI3K突变体和抑制剂对蛋白磷酸酶活性（PP1、PP2A等）（体内和体外）的调控，明确PI3K活性的作用。3)确定PI3K调控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，将对PP2A亚基和蛋白磷酸酶抑制剂（I-PP2As）的脂质结合或磷酸化对磷酸酶活性的调节进行深入研究。项目描述：b-肾上腺素能受体（bar）属于细胞表面受体中最大的家族，是心功能最强的调节剂之一。人类心力衰竭的特征是bar的下调（从细胞表面丢失）和慢性脱敏（受体无法接收信号）。虽然脱敏现象已经得到了很好的研究，但再敏（受体恢复活力并变得有能力发出信号的过程）还没有得到很好的理解。我们拨款提案中的研究将描述这种新机制。这一机制的阐明将导致分子的鉴定，这将使我们能够通过靶向再致敏来开发新的心力衰竭治疗策略。这是至关重要的，因为目前大多数治疗策略都涉及靶向受体来减弱下调。重要的是，确定重敏的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。