Mechanisms of Compartmentalized cAMP Signaling

描述(申请人提供)：通过环磷酸腺苷(CAMP)及其效应分子，如cAMP依赖的蛋白激酶(PKA)和cAMP激活的交换蛋白(EPAC)，调节多种细胞功能，包括细胞生长、增殖、代谢、存活和运动，以及胰岛素的分泌。细胞。我们研究的总体目标是阐明时空区划在cAMP信号中实现高特异性的分子机制和功能作用。CAMP信号通路的异常与肥胖和2型糖尿病等临床疾病有关，特别是在细胞功能方面。从机制上理解cAMP信号的特异性对于开发针对这些临床疾病的治疗策略至关重要。CAMP效应空间区划的概念是20年前提出的，但直到最近几年才出现了在细胞背景下研究cAMP信号的创新方法，以提供直接的机制证据。然而，尽管最近取得了这些进展，但对于cAMP及其效应物的时空调控机制，我们的理解仍然存在很大差距。此外，关于活动的时空模式中编码的信号信息如何转化为特定的功能反应，人们知之甚少。在我们的初步研究中，我们开发了用于监测EPAC作用的荧光生物传感器，用于跟踪多种信号活动的共成像方法，以及一种用于酶操纵亚细胞位置cAMP水平的方法。此外，我们最近的研究还发现了一个由cAMP、PKA和钙组成的振荡电路。细胞。在目前的提案中，基于这些初步发现，我们将专注于这些特定的目标来检验我们的中心假设，即cAMP、PKA和EPAC的活性在时空上是分开的，以具体调控这一途径的功能效应：1)进一步开发分子工具来测量cAMP、PKA和EPAC的动力学；2)阐明MIN6b细胞中振荡电路的调节机制和功能作用。

DESCRIPTION (provided by applicant): Signaling through cyclic AMP (cAMP) and its effector molecules, such as cAMP-dependent protein kinase (PKA) and exchange proteins activated by cAMP (Epac), regulates a variety of cellular functions including cell growth, proliferation, metabolism, survival and mobility, as well as insulin secretion in the case of pancreatic ? cells. The overall goal of our research is to elucidate the molecular mechanisms and functional roles of spatiotemporal compartmentalization in achieving high specificity in cAMP signaling. Aberrations in the cAMP signaling pathway have implications for clinical conditions such as obesity and type 2 diabetes mellitus, particularly in the context of ?-cell functions. A mechanistic understanding of cAMP signaling specificity is crucial to developing therapeutic strategies for these clinical conditions. The concept of spatial compartmentalization of cAMP effects was proposed 20 years ago, but only in recent years have innovative approaches to studying cAMP signaling in the cellular context become available to provide direct mechanistic evidence. However, despite these recent advances, there are still large gaps in our understanding about the mechanisms underlying the spatiotemporal regulation of cAMP and its effectors. Furthermore, little is known about how the signaling information encoded in the spatiotemporal patterns of activities is translated into specific functional responses. In our preliminary studies, we have developed fluorescent biosensors for monitoring Epac action, co-imaging approaches for tracking multiple signaling activities and a method for enzymatic manipulation of cAMP levels at subcellular locations. Furthermore, our recent studies have discovered an oscillatory circuit that consists of cAMP, PKA and Ca2+ in MIN6 ? cells. In the current proposal, building upon these preliminary findings, we will focus on these specific aims to test our central hypothesis that the activities of cAMP, PKA and Epac are spatiotemporally compartmentalized to specifically regulate functional effects of this pathway: 1) further develop molecular tools for measuring cAMP, PKA and Epac dynamics; 2) elucidate the regulatory mechanisms and functional roles of the oscillatory circuit in MIN6 b cells.