PKA targeting: A novel mechanism for GPCR resensitization

PKA 靶向：GPCR 再敏化的新机制

基本信息

批准号：
7751934
负责人：
SULEIMAN WAKIM BAHOUTH
金额：
$ 37万
依托单位：
UNIVERSITY OF TENNESSEE HEALTH SCI CTR
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-12-16 至 2012-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7751934
关键词：
A kinase anchoring protein AMPA Receptors Address Adrenergic Agents Adrenergic beta-Antagonists Affect Agonist Alanine Americas Amino Acids Arrestins Back Binding Binding Proteins Biological Assay Brain Cardiac Cardiac Myocytes Cells Complex Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cytoplasm DLG1 gene Data Figs - dietary G protein coupled receptor kinase G-Protein-Coupled Receptors G-substrate GTP-Binding Proteins Glutamates Goals Health Heart Heart Rate Heart failure Human Hypertension Individual Isoproterenol Knockout Mice Ligands Link Mediating Membrane Modification Molecular Mus Muscle Cells Mutagenesis Neonatal Opioid Performance Phosphorylation Phosphotransferases Process Protein Binding Protein Kinase Protein Kinase Interaction Proteins Rattus Receptors, Adrenergic, beta-1 Recycling Renin Role Signal Pathway Signal Transduction Site Sorting - Cell Movement Specificity Sympathetic Nervous System Tail Techniques Testing Transgenic Mice Ventricular Voltage-Gated Potassium Channel adrenergic beta-adrenergic receptor biological adaptation to stress desensitization fighting heart cell hypertension treatment imprint improved member novel novel therapeutics prevent public health relevance receptor receptor internalization receptor recycling response scaffold synapse-associated protein 97 tool

项目摘要

DESCRIPTION (provided by applicant): In the heart, beta-adrenergic receptor (¿-AR) stimulation increases contractile performance and heart rate as part of the 'fight-or-flight' stress response. Over stimulation of the sympathetic nervous system is a common theme in hypertension and is a primary contributor to heart failure. Increased sympathetic tone impinges on G protein-coupled receptors (GPCR) such as the ¿1- AR to promote their desensitization and internalization. The human ¿1-AR is internalized in response to ¿-agonists such as isoproterenol, followed by recycling (i.e. re-insertion back into the membrane) and resensitization of its signaling pathway. We determined that the recycling of the ¿1-AR is dependent on two domains one is serine312 in the third intracellular loop of the ¿1-AR, and the other is an "ESKV" sequence that corresponds to a type-1 PDZ ligand in the carboxy tail of the ¿1-AR. The ESKV sequence binds to a scaffold composed of the MAGUK protein SAP97, A-kinase anchoring protein 79 (AKAP79) and the cAMP-dependent protein kinase (PKA), that we have termed the ¿1-adrenergic receptosome. This scaffold is tethered to the ¿1-AR-PDZ via SAP97, which in turn binds to the AKAP79/PKA complex to assemble a trimeric scaffold (or receptosome) at the PDZ domain of the ¿1-AR. We have also shown that the components of this complex act in concert to facilitate the recycling of the agonist internalized ¿1-AR, through a common mechanism involving PKA-mediated phosphorylation of Ser312. Consequently, our primary hypothesis is, "that recycling and resensitization of the ¿1-AR is dependent upon the targeting of PKA to the ¿1-AR via the ¿1-adrenergic receptosome". We propose to determine the domains within each member of the ¿1-adrenergic receptosome that are functionally involved in recycling and resensitization of the ¿1-AR in HEK-293 cells and rat/mouse neonatal ventricular myocytes. In aims #1 and 2, we will identify the specific domains in AKAP79 and in SAP97, respectively that facilitate the recycling and resensitization of the ¿1- AR. Then we will determine if AKAP79 and/or SAP97 are essential for recycling of the ¿1-AR in cardiac ventricular myocytes prepared from neonatal rats or knockout mice. Finally in aim #3: we will characterize the molecular determinants of the PDZ motif of the ¿1-AR that regulate its binding to SAP97 and facilitate the recycling and resensitization of the ¿1-AR in HEK-293 and in heart cells. Results of these studies will provide new information concerning the mechanisms of ¿1-AR resensitization that may ultimately provide new therapeutic paradigms to improve ¿-blocker therapy, which is a main tool for the treatment of hypertension and heart failure. PUBLIC HEALTH RELEVANCE: In the heart, beta-1-adrenergic receptor stimulation increases contractile performance and heart rate as part of the 'fight-or-flight' stress response. New information indicates that the beta-1-adrenergic receptor is connected to other cellular networks that affect its fate and markedly broaden the range of its actions within the cell. By investigating the function of these networks, we believe that we can improve on the performance of beta-blockers, which are widely-used in treating two major health problems in America, namely hypertension and heart failure.

描述（由申请人提供）：在心脏中，β-肾上腺素能受体（¿-AR）刺激会增加收缩性能和心率，作为“战斗或逃跑”应激反应的一部分，交感神经系统的过度刺激是一种现象。高血压的常见主题，是导致心力衰竭的主要原因，交感神经紧张会影响 G 蛋白偶联受体 (GPCR)，例如 ¿ 1- AR促进他们的脱敏和内化。 1-AR 内化以响应 ¿ -激动剂，例如异丙肾上腺素，然后进行回收（即重新插入膜中）并使其信号通路重新敏化，我们确定了 ¿ 1-AR 依赖于两个结构域，其中一个是 ¿ 的第三个细胞内环中的丝氨酸 312 1-AR，另一个是“ESKV”序列，对应于 ¿ 羧基尾中的 1 型 PDZ 配体1-AR。ESKV 序列与由 MAGUK 蛋白 SAP97、A-激酶锚定蛋白 79 (AKAP79) 和 cAMP 依赖性蛋白激酶 (PKA) 组成的支架结合，我们将其称为 ¿ 1-肾上腺素受体体连接到 ¿ 1-AR-PDZ 通过 SAP97，进而与 AKAP79/PKA 复合物结合，在 ¿ 的 PDZ 结构域处组装三聚体支架（或受体体） 1-AR。我们还表明，该复合物的成分协同作用，促进内化激动剂的回收。 1-AR，通过涉及 PKA 介导的 Ser312 磷酸化的常见机制，我们的主要假设是，“1-AR 的回收和再敏化取决于 PKA 通过 1-AR 靶向 1-AR。 1-肾上腺素受体体”。我们建议确定 ¿ 每个成员内的域1-肾上腺素受体体在功能上参与 ¿ 的回收和再敏化HEK-293 细胞和大鼠/小鼠新生儿心室肌细胞中的 1-AR 在目标#1 和 2 中，我们将分别确定 AKAP79 和 SAP97 中促进 ¿ 的回收和再敏化的特定结构域。 1- AR. 然后我们将确定 AKAP79 和/或 SAP97 是否对于回收 ¿新生大鼠或基因敲除小鼠制备的心室肌细胞中的 1-AR 最后，目标#3：我们将表征 ¿ 的 PDZ 基序的分子决定因素。 1-AR 调节其与 SAP97 的结合并促进 ¿ HEK-293 和心脏细胞中的 1-AR 这些研究结果将提供有关 ¿ 机制的新信息。 1-AR 再敏化可能最终提供新的治疗范例来改善 ¿ - 阻滞剂疗法，这是治疗高血压和心力衰竭的主要工具。公共卫生相关性：在心脏中，β-1-肾上腺素能受体刺激可增加收缩性能和心率，作为“战斗或逃跑”的一部分。新的信息表明，β1-肾上腺素能受体与影响其命运的其他细胞网络相连，并显着扩大了其在细胞内的作用范围，我们相信我们可以改善这些网络的功能。 β-受体阻滞剂的性能，在美国，β-受体阻滞剂广泛用于治疗两大健康问题，即高血压和心力衰竭。