Regulation of Beta Adrenergic Receptor Function by localized P13K Inhibition

通过局部 P13K 抑制调节 β 肾上腺素能受体功能

基本信息

批准号：
7492819
负责人：
Howard A Rockman
金额：
$ 40.83万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7492819
关键词：
1-Phosphatidylinositol 4-Kinase Adenylate Cyclase Agonist Animal Model Binding Calcineurin Calcineurin inhibitor Calcium Calsequestrin Cardiac Cardiac Myocytes Catecholamines Cell Nucleus Cells Chronic Clathrin Complex Constriction procedure Correlative Study Coupling Data Dominant-Negative Mutation Down-Regulation Endocytosis Evaluation Event Exercise Failure Family suidae GTP-Binding Proteins Gene Expression Gene Transfer Generations Heart Heart Hypertrophy Heart failure Human Hypertrophy In Vitro Indium Infarction Knock-out Knowledge Lead Lipids Localized Mediating Modeling Molecular Mus Muscle Cells NF-AT Numbers Oryctolagus cuniculus Pathogenesis Peptides Performance Phenotype Phosphatidylinositols Phosphorylation Phosphotransferases Physiological Pik-off Play Process Protein Kinase Protein Overexpression Proteins Protocols documentation Regulation Rodent Model Role Signal Pathway Signal Transduction Stimulus Sus scrofa TFAP2A gene TRP channel TRPC3 ion channel Tertiary Protein Structure Testing Transactivation Transgenes Transgenic Animals Transgenic Mice adenoviral-mediated beta-adrenergic receptor beta-arrestin coated pit desensitization gain of function in vivo loss of function mouse model muscle LIM protein mutant nuclear factors of activated T-cells pressure prevent promoter receptor receptor function receptor internalization response transcription factor

项目摘要

Beta-adrenergic receptor (betaAR) signaling plays an important role in the regulation of cardiac performance. BetaAR downregulation and desensitization resulting in reduced responsiveness to catecholamines are hallmarks of the failing heart. In human heart failure, diminished receptor number (downregulation) and impaired coupling (desensitization) of betaARs result in reduced responsiveness to catecholamines. The process that initiates desensitization of the receptor begins with the phosphorylation of betaARs by beta-adrenergic receptor kinase1 (betaARK1), followed by the binding of beta-arrestin, which together inhibits coupling to G protein and loss of effector (adenylyl cyclase) signaling. The beta-arrestin receptor complex is then targeted to the clathrin-coated pits for endocytosis through a process involving interaction of beta-arrestin with AP-2 and clathrin. Recently we have shown that betaARK1 interacts with the lipid kinase, phosphoinositide 3-kinase (PI3K), to form a cytosolic complex. We have shown that the betaARK/PI3K interaction is critical for the agonist-mediated internalization of betaARs and if the betaARK/PI3K interaction is disrupted in vivo, betaARs do not undergo downregulation and desensitization with chronic catecholamine stimulation. Importantly, PI3K contains both lipid kinase and protein kinase activity. Although we have shown that the lipid products of PI3K are important for receptor internalization, it is not yet known whether the protein kinase activity of PI3K is necessary for betaAR endocytosis and activation of downstream signaling pathways. Since receptor-localized PI3K plays a critical role in betaAR internalization, knowledge of the distinct roles of the protein and lipid kinase activities in the process of betaAR sequestration, resensitization and signaling would contribute to a better understanding of the regulation of betaAR function in the pathogenesis of heart failure. Our Central Hypothesis is that downregulation of cardiac betaARs, in vivo, can be prevented through inhibition of the local generation of D-3 phosphatidylinositols by PI3K within the receptor complex and preserving betaAR function in the failing heart will lead to an amelioration of heart failure in both small and large animal models of cardiac failure. We will test the hypothesis that the protein kinase activity of PI3K is required for betaAR internalization and downregulation, and that betaAR function will be normal in hearts displaying a "physiological" hypertrophic phenotype compared to hearts that show a "pathological" hypertrophic phenotype. Specific Aims (1) To determine the molecular mechanisms of PI3K-mediated betaAR internalization by studying PI3K mutants that lack either protein kinase activity or lipid kinase activity; (2) To determine whether overexpression of the minimal peptide of PI3K (PIK domain peptide, PIKdp), which acts as a dominant negative in vitro, will prevent betaAR downregulation in vivo and rescue mouse models of heart failure: a) chronic pressure overload (transverse aortic constriction, TAC), b) Calsequestrin overexpression X PIKdp, c) muscle LIM protein knock out X PIKdp; (3) To determine whether overexpression of PI3Kdp in vivo by adenoviral-mediated gene transfer to the heart will prevent betaAR downregulation and rescue two large animal models of cardiac failure: a) AdenoPIKdp injected into a rabbit infarct model, b) the pacing pig heart failure model; (4) To determine whether the mechanism that causes transition from hypertrophy to heart failure is due to the type of load on the heart (exercise vs. pressure) or the chronicity of the load. Chronic TAC will be compared to intermittent TAC and two exercise protocols in mice, together with a comprehensive evaluation of the developing cardiac phenotype.

β-肾上腺素能受体 (betaAR) 信号在心脏功能的调节中发挥着重要作用。 BetaAR 下调和脱敏导致对儿茶酚胺的反应性降低，这是心脏衰竭的标志。在人类心力衰竭中，βAR 受体数量减少（下调）和偶联受损（脱敏）会导致对儿茶酚胺的反应性降低。启动受体脱敏的过程始于 β-肾上腺素能受体激酶 1 (betaARK1) 对 βAR 的磷酸化，然后与 β-arrestin 结合，共同抑制与 G 蛋白的偶联和效应器（腺苷酸环化酶）信号传导的丧失。然后，通过涉及 β-抑制蛋白与 AP-2 和网格蛋白相互作用的过程，β-抑制蛋白受体复合物被靶向网格蛋白包被的凹坑进行内吞作用。最近我们发现 betaARK1 与脂质激酶、磷酸肌醇 3-激酶 (PI3K) 相互作用，形成胞质复合物。我们已经证明 betaARK/PI3K 相互作用对于激动剂介导的 betaAR 内化至关重要，并且如果 betaARK/PI3K 相互作用在体内被破坏，betaAR 不会因慢性儿茶酚胺刺激而发生下调和脱敏。重要的是，PI3K 含有脂质激酶和蛋白激酶活性。尽管我们已经证明 PI3K 的脂质产物对于受体内化很重要，但尚不清楚 PI3K 的蛋白激酶活性是否是 betaAR 内吞作用和下游信号通路激活所必需的。由于受体定位的 PI3K 在 betaAR 内化中起着至关重要的作用，因此了解蛋白质和脂质激酶活性在βAR隔离、再敏化和信号转导过程中的独特作用将有助于更好地了解βAR功能在心力衰竭发病机制中的调节。我们的中心假设是，体内心脏βAR的下调可以通过受体复合物内PI3K抑制D-3磷脂酰肌醇的局部生成来预防，并且在衰竭心脏中保留βAR功能将导致心力衰竭的改善小型和大型动物心力衰竭模型。我们将测试以下假设：PI3K 的蛋白激酶活性是 betaAR 内化和下调所必需的，并且与显示“病理性”肥厚表型的心脏相比，在显示“生理性”肥厚表型的心脏中 betaAR 功能将正常。具体目的 (1) 通过研究缺乏蛋白激酶活性或脂质激酶活性的 PI3K 突变体，确定 PI3K 介导的 betaAR 内化的分子机制； (2) 确定PI3K最小肽（PIK结构域肽，PIKdp）是否过度表达，其作为显性失活在体外，将防止体内 betaAR 下调并挽救心力衰竭小鼠模型：a) 慢性压力超负荷（横主动脉缩窄，TAC），b) Calsequestrin 过度表达 X PIKdp，c) 肌肉 LIM 蛋白敲除 X PIKdp； (3) 确定通过腺病毒介导的基因转移至心脏而在体内过度表达 PI3Kdp 是否会阻止 betaAR 下调并挽救两种大型动物心力衰竭模型：a) 将 AdenoPIKdp 注射到兔梗塞模型中，b) 起搏猪心脏失效模型； (4) 确定导致从肥大转变为心力衰竭的机制是否是由于心脏负荷类型（运动与压力）或负荷的长期性所致。将慢性 TAC 与间歇性 TAC 和小鼠的两种运动方案进行比较，并对发育中的心脏表型进行综合评估。