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。 在人类心力衰竭中，与受损的G蛋白偶联（脱敏）相关的质膜上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（bARK 1）对激动剂激活的bAR进行磷酸化而启动的。β-抑制蛋白与磷酸化受体结合，导致效应物（腺苷酸环化酶）信号传导的丧失。 bAR复合物靶向内吞作用，导致内体区室中受体的去磷酸化，然后再循环回质膜。 以前，我们已经表明，bARK 1与磷脂酰肌醇3-激酶（PI 3 K）相互作用，形成一个细胞质复合物，靶向PI 3 K的活化受体，其中PI 3 K在受体内吞作用中发挥作用。 我们现在已经发现了一种新的现象，受体在质膜在体内小鼠心脏过表达的非活性PI 3 K突变体。 此外，我们已经证明，bAR再敏化是有益的，因为它通过保存bAR功能来防止有害的心脏重塑。 这些初步数据与目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到质膜之前必须经历内化以脱磷酸化。 调节这种质膜受体再敏感化的新现象的分子机制尚不清楚。 我们假设受体靶向的PI 3 K活性负调节质膜上的受体再敏化。 因此，抑制受体定位的PI 3 K活性导致质膜处的受体再敏化，而不需要内化。 本研究的具体目的是：1）确定PI 3 K活性是否调节bAR再敏感。将使用含有蛋白质或/和脂质激酶活性的PI 3 K突变体，通过G蛋白偶联、受体磷酸化、腺苷酸环化酶活性/cAMP水平对受体再敏化进行详细分析。 2)描述PI 3 K介导的bAR再敏感是否通过调节蛋白磷酸酶活性发生。 蛋白磷酸酶（PP 1、PP 2A等）活性调节分析(in体内和体外）通过PI 3 K突变体和抑制剂来进行，以确定PI 3 K活性的作用。3)目的：探讨PI 3 K调节PP 2A活性的分子机制。 通过脂质结合研究、代谢标记、RNAi敲除和使用PP 2A和I2-PP 2a突变体，将对PP 2A亚基和蛋白磷酸酶抑制剂（I-PP 2A）的脂质结合或磷酸化调节磷酸酶活性进行深入研究。项目叙述：β-肾上腺素能受体（bAR）属于细胞表面受体的最大家族，并且是心脏功能的最强调节剂之一。 人心力衰竭的特征在于bAR的下调（从细胞表面损失）和慢性脱敏（受体不能传递信号）。 虽然脱敏现象得到了很好的研究，但再敏化（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。 在我们的拨款申请中的研究将描述这种新的机制。 阐明这一机制将导致识别的分子，使我们能够开发新的治疗策略，心力衰竭的靶向脱敏。 这是至关重要的，因为大多数目前的治疗策略涉及靶向受体以减弱下调。 重要的是，确定再致敏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。