Mechano-Chemical Regulation of GPCR/PKA Signaling During Cell Migration

 描述(由申请人提供)：细胞与其微环境保持着张力的动态平衡，这种“机械顺应性”控制着多种细胞功能，包括细胞的命运、形状和运动。尽管这一点很重要，但细胞感知和响应细胞外基质的机械性质的分子机制还没有完全了解。我们的实验室证实，cAMP依赖的蛋白激酶(PKA)在细胞的前沿被丰富和激活，这种定位对细胞迁移非常重要。在我们正在努力阐明细胞迁移过程中PKA的空间调节机制的过程中，我们最近发现，局部的PKA活性受细胞张力的调节。具体地说，一旦肌动球蛋白收缩能力被抑制，前沿PKA活性就会迅速丧失。此外，当细胞被机械拉伸时，PKA以一种依赖于张力的方式在拉伸方向上被迅速和局部激活。最后，抑制PKA也阻止了细胞外基质刚性和细胞-基质张力梯度引导的趋性迁移。我们目前的重点是了解细胞张力与PKA局部调控的联系机制。最近的初步数据证实，细胞的收缩能力和PKA的局部激活都依赖于细胞外钙通过拉伸激活通道TRPM7的内流。更多的数据有力地表明，G蛋白偶联受体-A2B腺苷受体(ADORA2B)-也在这一机制中发挥重要作用。根据我们的观察，我们假设迁移细胞前沿的PKA的局部激活是由一种机械力化学机制调节的，该机制涉及细胞张力的局部增加、细胞外钙的内流和ADORA2B的激活之间的相互作用。我们将通过确定ADORA2B在细胞迁移过程中PKA的机械激活中的作用，描述ADORA2B在细胞迁移过程中的机械力化学激活/调节机制，以及确定收缩性、钙和ADORA2B在调节局部PKA活性和细胞迁移中的机制层次来验证这一假说。在拟议的研究结束时，我们将建立一条典型的途径，在该途径中，细胞力学与GPCR信号级联的局部耦合调节细胞骨架的动力学和细胞的运动。

 DESCRIPTION (provided by applicant): Cells maintain a dynamic equilibrium of tension with their microenvironment and this `mechanoreciprocity' controls a wide variety of cellular functions, including cell fate, shape, and movement. Despite this importance, the molecular mechanisms through which cells sense and respond to the mechanical nature of the extracellular matrix are not completely understood. Our laboratory established that the cAMP-dependent protein kinase (PKA) is enriched and activated in the leading edge of cells and that this localization is important for cell migration. In our ongoing efforts to elucidate the mechanis for the spatial regulation of PKA during cell migration, we have recently found that localized PKA activity is regulated by cellular tension. Specifically, leading edge PKA activity is rapidly lost upon inhibition of actomyosin contractility. Moreover, when cells are mechanically stretched, PKA is rapidly and locally activated - in a tension-dependent manner - in the direction of stretch. Finally, inhibition of PKA also blocks durotaxis - cell migration guided by gradients in ECM rigidity and cell-matrix tension. Our current focus is to understand the mechanism that couples cellular tension to localized regulation of PKA. Recent preliminary data establishes that both cellular contractility and localized activation of PKA are dependent on influx of extracellula Ca2+ via the stretch-activated channel TRPM7. Additional data strongly suggest that a G- protein coupled receptor - the A2B adenosine receptor (ADORA2B) - also plays an important role in this mechanism. Based on our observations, we hypothesize that localized activation of PKA in the leading edge of migrating cells is regulated by a mechano-chemical mechanism involving interplay between localized increases in cellular tension, influx of extracellular Ca2+, and activation of ADORA2B. We will test this hypothesis by determining the role of ADORA2B in mechanical activation of PKA during cell migration, delineating the mechanism of mechano-chemical activation/regulation of ADORA2B during cell migration, and determining the mechanistic hierarchy of contractility, Ca2+, and ADORA2B in regulating localized PKA activity and cell migration. At the end of the proposed studies, we will have established a paradigmatic pathway in which localized coupling of cellular mechanics to a GPCR signaling cascade regulates cytoskeletal dynamics and cell motility.