Mechanisms of cAMP Compartmentation

描述(申请人提供)：第二信使cAMP参与调节我们体内几乎每个细胞的各种反应。一个例子是心肌细胞，在那里，β-肾上腺素能受体通过产生cAMP来介导交感神经对电、机械和代谢活动的影响。然而，实际上有多种受体能够调节心肌细胞中cAMP的产生，就像许多其他细胞一样。尽管不同的受体可能共享这个共同的第二信使，但它们并不都能引发相同的功能反应。通常的解释是，cAMP的产生是分区的，在空间上限制了某些受体产生的反应的程度。受体的激活不会导致整个细胞内cAMP产生的统一刺激或抑制，这一想法可能直观地看起来是显而易见的，但它是如何实现的还没有完全理解。通常的假设是，磷酸二酯酶作为扩散的功能障碍，创建离散的cAMP信号域。我们将检验这一假设，即磷酸二酯酶活性在分隔中起重要作用，但不是扩散的障碍。我们还将测试细胞骨架和依赖胆固醇的质膜组织在这一过程中发挥关键作用的假设。这一应用的优势在于将用于解决这些假设的方法的创新组合。我们将采用一种系统生物学的方法，包括使用基于多重荧光共振能量转移(FRET)的生物传感器来监测活细胞不同亚细胞隔间中的cAMP活性，以及对分隔的cAMP信号进行定量计算建模。CAMP的产生是引起多种细胞功能有益改变的重要手段。然而，cAMP依赖的信号也可以在正确(或错误)的条件下产生病理反应。有人假设，生理和病理效应之间的差异可能是cAMP信号适当区隔的功能。因此，了解在亚细胞水平上负责协调cAMP时空分布的因素的作用，对于开发新的策略来预防或治疗与许多不同疾病状态相关的不利反应可能是重要的。

DESCRIPTION (provided by applicant): The second messenger cAMP is involved in regulating a variety of responses in virtually every cell in our bodies. One example is the cardiac myocyte, where beta-adrenergic receptors mediate sympathetic effects on electrical, mechanical, and metabolic activity through the production of cAMP. However, there are actually multiple receptors capable of regulating cAMP production in cardiac myocytes, just like many other cells. Even though various receptors may share this common second messenger, they do not all elicit the same functional responses. The common explanation is that cAMP production is compartmentalized, spatially limiting the extent of responses produced by certain receptors. The idea that activation of a receptor does not lead to uniform stimulation or inhibition of cAMP production throughout the cell might seem intuitively obvious, yet it is not fully understood how this is achieved. The common assumption is that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We will test the hypothesis that phosphodiesterase activity plays an important role in compartmentation, but not as a barrier to diffusion. We will also test the hypothesis that the cytoskeleton and cholesterol-dependent organization of the plasma membrane play essential roles in this process. The strength of this application lies in the innovative combination of methods that will be used to address these hypotheses. We will employ a systems biology approach that involves the use of multiple fluorescence resonance energy transfer (FRET) based biosensors to monitor cAMP activity in distinct subcellular compartments of live cells, together with quantitative computational modeling of compartmentalized cAMP signaling. The production of cAMP is an important means of eliciting beneficial changes in function of many cell types. However, cAMP-dependent signaling can also produce pathological responses under the right (or wrong) conditions. It has been hypothesized that the difference between the physiological and pathological effects may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and temporal distribution of cAMP at the subcellular level could be important for developing new strategies for the prevention or treatment of unfavorable responses associated with many different disease states.