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: 8236869
• 负责人: Sathyamangla V Prasad
• 金额: $ 38.24万
• 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 2013-12-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8236869
• 关键词: 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）信号传导是心脏功能最强大的调节剂之一。 在人体心力衰竭中，与G蛋白偶联受损（脱敏）相关的质膜的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Bar激酶-1（Bark1）激活的棒磷酸化引发的。 B- arrestin与磷酸化受体结合，导致效应子（腺苷酸环化酶）信号传导的丧失。 棒络合物的靶向内吞作用，导致内体隔室中受体去磷酸化，然后再回收回到质膜。 以前，我们已经表明，Bark1与磷酸肌醇3-激酶（PI3K）相互作用，形成靶向PI3K与活化受体的胞质复合物，其中PI3K在受体内吞作用中起着作用。 现在，我们通过不活跃的PI3K突变体的心脏过表达在小鼠体内的质膜中发现了一种新型的受体膜化现象。 此外，我们已经证明了钢筋汇总化是有益的，因为它可以通过保留条函数来防止有害的心脏重塑。 这些初步数据与当前受体复敏的范式相反，该范例表明，磷酸化脱敏的受体必须在回收回到质膜之前要进行内在化。 调节这种新型质膜受体敏感性现象的分子机制尚不清楚。 我们假设受体靶向PI3K活性对质膜上的受体敏化负面调节。 因此，抑制受体局部PI3K活性会导致受体在质膜上的受体敏化，而无需内在化。 在本研究中提出了以下特定目的：1）确定PI3K活性是否调节棒函数化。通过使用含有蛋白质或/和脂质激酶活性的PI3K突变体进行详细的分析受体偶联，受体磷酸化，腺苷酸环化酶活性/cAMP水平的受体脱敏化分析。 2）描述PI3K介导的BAR介绍是否通过调节蛋白质磷酸酶活性而发生。 将通过PI3K突变体和抑制剂来定义PI3K活性的作用，对PI3K突变体和抑制剂的调节（PP1，PP2A等）调节（体内和体外）。 3）确定PI3K调节PP2A活性的基本机制。 通过脂质结合研究，代谢标记，RNAI敲低以及PP2A和I2-PPP2A突变剂的使用，将对PP2A亚基和蛋白质磷酸酶（I-PP2AS）的脂质结合或磷酸化的调节进行深入研究。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。 人体心力衰竭的特征是下调（细胞表面丧失）和慢性脱敏（受体对信号的无能）。 虽然对脱敏现象进行了充分的研究，但尚未很好地理解脱敏的现象（受体恢复活力并胜任信号的过程）。 我们的赠款提案中的研究将描述这种新型机制。 阐明这种机制将导致鉴定分子，这将使我们能够通过靶向敏化来开发新的治疗性心力衰竭治疗策略。 这至关重要，因为当前的大多数治疗策略涉及针对受体减轻下调的靶向。 重要的是，确定敏化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。