Nuclear alpha1-Adrenergic Receptor Signaling in Adult Mouse Cardiace Myocytes

描述（由申请人提供）：本研究的目的是确定成年小鼠心肌细胞中a1-肾上腺素能受体（a1-AR）存活信号的分子机制。最近的证据表明，a1- ar是心脏正常生长发育和心肌适应应激所必需的。此外，使用a1-AR拮抗剂或a1受体阻滞剂的临床试验导致心力衰竭的显著增加。本实验室正在进行的研究表明，先前描述的a1A-AR-ERK存活信号是由位于核膜上的a1A-AR启动的，当a1A-AR被激活时，会诱导磷酸化的ERK在质膜的小泡中积累。这种信号通路是独特的，因为它不符合G蛋白偶联受体信号的经典模型，并提出了成人心肌细胞中a1 a - ar - erk存活信号的令人兴奋的模型。我们假设激活的核a1-AR通过Gaq和PLCP1启动信号传导，导致核内PKC激活，PKC转运到质膜的小泡中，激活ERK上游已知的激酶途径（Ras， Raf和MEK）。此外，我们假设caveolin-3是a1-AR诱导的ERK信号的调节因子，Bad和/或fox01是a1-AR-ERK生存信号的下游靶点。我们将通过确定参与a1-AR存活信号从细胞核转导到质膜的信号蛋白、小窝蛋白-3对存活信号的调节作用以及磷酸化ERK的下游靶标来验证这些假设。为实现这些目标，我提出以下具体目标：目的1：确定caveolin-3如何调节成年小鼠心肌细胞中a1A-ERK介导的存活信号和活化ERK的下游靶点。目的2：确定成年小鼠心肌细胞活化核a1-AR导致质膜ERK活化的信号转导途径。我们的目标是确定成人心肌细胞a1-AR-ERK存活信号背后的分子机制。采用ERK磷酸化和细胞死亡的Western blot方法，对分离的具有不同表达caveolin-3的心肌细胞进行检测，以确定caveolin-3的调节作用。利用Western blot分析Bad和fox01的磷酸化状态，我们将确定这两种蛋白是否是活化ERK的下游靶点。免疫细胞化学、PKC同工酶抑制和ERK磷酸化的Western blot分析将用于确定a1-AR存活信号如何到达质膜的小泡。

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.