Role of the mAKAP Complex in Cardiac Hypertrophy

描述（由申请人提供）：由a激酶锚定蛋白（AKAPs）形成的称为“信号体”的多分子复合物有助于第二信使cAMP在空间和时间上限制细胞内信号传导。靶向单个信号体中存在的独特蛋白质-蛋白质相互作用可能构成一种新的药物发现方法，产生一类新的选择性心脏治疗方法，显示最小的脱靶副作用。其中一个信号体是由mAKAPb组织的，mAKAPb是一种支架蛋白，结合腺苷酸环化酶5、camp依赖性酶蛋白激酶a和Epac1以及camp特异性磷酸二酯酶PDE4D3。通过包含cAMP合成、降解和功能所需的所有酶，mAKAPb复合物可以自主调节和响应局部控制的cAMP水平。mAKAPb信号小体还含有ERK5丝裂原活化蛋白激酶和Ca2???？因此，肾上腺素能和gp130细胞因子/白血病抑制因子受体在体外诱导新生儿肌细胞肥大的作用被表达mAKAPb的RNAi破坏。该应用有三个特定目的，以解决两个中心假设：(1)mAKAPb在体内心脏重塑的调节中起关键作用；(2)mAKAPb信号体形成一个自主的cAMP信号室，其破坏将导致局部cAMP水平和整体心肌细胞表型的变化。特异性目的1:mAKAPb在心脏重塑中的作用。研究人员将使用一种新的“floxed”mAKAP等位基因在小鼠中测试mAKAPb支架与病理性重塑的体内相关性。将使用心脏特异性cre转基因来删除mAKAP基因，并对非应激、衰老小鼠和慢性异丙肾上腺素输注、主动脉横缩和心肌梗死小鼠进行研究。特异性目标2:mAKAPb复合物对AC5的调控。AC5直接与mAKAPb的n端结构域结合。我们将在体外和体内利用一种新的转基因小鼠研究AC5结合肽如何通过结合mAKAPb来调节AC5的活性，这种转基因小鼠在心脏中有条件地表达AC5结合肽。特异性目标3：通过mAKAPb信号体控制局部cAMP水平。将通过与mAKAPb融合的cAMP FRET传感器在培养的成人和新生儿心肌细胞中的表达来研究mAKAPb信号体对活细胞中局部cAMP水平的调节。使用野生型mAKAPb融合传感器获得的信号将与使用缺乏单个结合伙伴结合位点的mAKAPb传感器获得的信号进行比较，从而揭示单个支架蛋白复合物的破坏如何影响细胞内信号传导。通过这些特定目的获得的数据应该可以确定mAKAPb信号体是肥厚性信号网络中的一个重要节点，并且可以作为特异性药物治疗不适应重构和预防心力衰竭的候选靶点。

DESCRIPTION (provided by applicant): The formation of multimolecular complexes called "signalosomes" by A-kinase anchoring proteins (AKAPs) contributes to the spatial and temporal restriction of intracellular signaling by the second messenger cAMP. Targeting unique protein-protein interactions present within individual signalosomes may constitute a novel approach to drug discovery, yielding a new class of selective cardiac therapies displaying minimal off- target side-effects. One such signalosome is organized by mAKAPb, a scaffold protein that binds adenylyl cyclase 5, the cAMP-dependent enzymes protein kinase A and Epac1, and the cAMP-specific phosphodiesterase PDE4D3. By including all of the enzymes necessary for cAMP synthesis, degradation, and function, mAKAPb complexes may autonomously regulate and respond to locally controlled cAMP levels. mAKAPb signalosomes also contain ERK5 mitogen-activated protein kinase and the Ca2???? dependent phosphatase calcineurin Ab. Accordingly, the induction of neonatal myocyte hypertrophy in vitro by adrenergic and gp130 cytokine/leukemia inhibitory factor receptors is impaired by RNAi of mAKAPb expression. This application has three Specific Aims that address two central hypotheses: (1) that mAKAPb plays a critical role in the regulation of cardiac remodeling in vivo, and (2) that the mAKAPb signalosome forms an autonomous cAMP signaling compartment whose disruption will result in changes both in local cAMP levels and overall myocyte phenotype. Specific Aim 1: The role of mAKAPb in cardiac remodeling. The in vivo relevance of the mAKAPb scaffold to pathologic remodeling will be tested in mice using a new "floxed" mAKAP allele. The mAKAP gene will be deleted using a cardiac-specific cre transgene, and both unstressed, aging mice and mice subjected to chronic isoproterenol infusion, transverse aortic constriction and myocardial infarction will be studied. Specific Aim 2: Regulation of AC5 by mAKAPb Complexes. AC5 directly binds to a N-terminal domain in mAKAPb. How AC5 activity is regulated by binding mAKAPb will be investigated in vitro and in vivo using a novel transgenic mouse in which an AC5-binding peptide is conditionally expressed in the heart. Specific Aim 3: Control of local cAMP levels by the mAKAPb signalosome. mAKAPb signalosome regulation of local cAMP levels in living cells will be investigated by the expression in cultured adult and neonatal cardiac myocytes of cAMP FRET sensors fused to mAKAPb. Signals obtained with a wildtype mAKAPb fusion sensor will be compared to that obtained using mAKAPb sensors lacking binding sites for individual binding partners, thereby revealing how the disruption of an individual scaffold protein complex affects intracellular signaling. Data obtained by these Specific Aims should establish the mAKAPb signalosome as an important node in the hypertrophic signaling network and as a candidate target for specific drug therapy for maladaptive remodeling and the prevention of heart failure.