Nuclear alpha1-Adrenergic Receptor Signaling in Adult Mouse Cardiace Myocytes

描述(申请人提供)：本研究的目的是确定A1-肾上腺素能受体(A1-AR)存活信号在成年小鼠心肌细胞中的分子机制。最近的证据表明，A1-ARs对于心脏的正常生长发育和心肌对应激的适应都是必需的。此外，A1-AR拮抗剂或A1-阻滞剂的临床试验导致心力衰竭显著增加。本实验室正在进行的研究表明，先前描述的A1A-AR-ERK生存信号是由定位在核膜上的A1A-AR启动的，当激活时，诱导磷酸化的ERK在质膜上的小窝中积聚。这条信号通路是独特的，因为它不符合G蛋白偶联受体信号的经典模型，而是成人心肌细胞A1 A-AR-ERK生存信号的激发模型。我们推测，激活的核A1-AR通过Gaq和PLCP1启动信号，导致核内PKC的激活，PKC转位到质膜上的小凹，并激活ERK上游的已知激酶途径(RAS、Raf和MEK)。此外，我们假设小窝蛋白-3是A1-AR诱导的ERK信号的调节因子，Bad和/或FOXO1是A1-AR-ERK生存信号的下游靶点。我们将通过确定参与将A1-AR生存信号从细胞核传递到质膜的信号蛋白，小窝蛋白-3对生存信号可能有什么调节作用，以及磷酸化ERK的下游靶点是什么来检验这些假说。为了实现这些目标，我提出了以下具体目标。目的1：研究小窝蛋白-3对A1a-ERK介导的成年小鼠心肌细胞存活信号及激活ERK下游靶点的调节作用。目的：研究成年小鼠心肌细胞胞核A1-AR活化导致质膜ERK活化的信号转导途径。我们的目标是确定A1-AR-ERK存活信号在成人心肌细胞中的分子机制。对不同表达小窝蛋白-3的分离心肌细胞进行ERK磷酸化的蛋白质印迹和细胞死亡分析，将确定小窝蛋白-3的调节作用。通过蛋白质印迹分析Bad和FOXO1的磷酸化状态，我们将确定这两种蛋白是否都是ERK激活的下游靶点。免疫细胞化学、PKC同工酶抑制和ERK磷酸化的Western印迹分析将用于确定A1-AR生存信号如何到达质膜上的小凹。

DESCRIPTION (provided by applicant): The objective of this research is to define the molecular mechanisms of a1-adrenergic receptor (a1-AR) survival signaling in adult mouse cardiac myocytes. Recent evidence suggests that a1-ARs are required for normal growth and development of the heart and myocardial adaptation to stress. Additionally, clinical trials with a1-AR antagonists, or a1-blockers, led to significant increases in heart failure. Ongoing research in this lab has revealed that the previously described a1A-AR-ERK survival signal is initiated by a1A-AR localized on the nuclear membrane that, when activated, induce accumulation of phosphorylated ERK in caveolae at the plasma membrane. This signaling pathway is unique because it does not fit the classical models of G- protein coupled receptor signaling and presents a provocative model for a1 A-AR-ERK survival signaling in adult cardiac myocytes. We hypothesize that activated nuclear a1-AR initiate signaling thru Gaq and PLCP1 leading to activation of PKC within the nucleus, PKC translocates to caveolae at the plasma membrane and activates the known kinase pathway (Ras, Raf, and MEK) upstream of ERK. Furthermore we hypothesize that caveolin-3 is a regulator of a1-AR induced ERK signaling, and that Bad and/or FOXO1 are downstream targets of a1-AR-ERK survival signal. We will test these hypotheses by determining the signaling proteins involved in transducing the a1-AR survival signal from the nucleus to the plasma membrane, what regulatory role caveolin-3 might have on the survival signal, and what the down stream targets of phosphorylated ERK is/are. I propose the following specific aims to achieve these goals. Aim 1: Determine how caveolin-3 regulates a1A-ERK mediated survival signaling and the downstream targets of activated ERK in adult mouse cardiac myocytes. Aim 2: Determine the signal transduction pathway from activated nuclear a1-AR that leads to activated ERK at the plasma membrane in adult mouse cardiac myocytes. Our goal is to determine the molecular mechanisms behind a1-AR-ERK survival signal in adult cardiac myocytes. Using Western blot of ERK phosphorylation and cell death assays on isolated cardiac myocytes with varying expression of caveolin-3 will determine the regulatory role of caveolin-3. Using Western blot analysis of the phosphorylation status of Bad and FOXO1 we will determine if either protein is a downstream target of activated ERK. Immunocytochemistry, inhibition of PKC isozymes and Western blot analysis of ERK phosphorylation will be used to determine how the a1-AR survival signal reaches caveolae at the plasma membrane.