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张力的动态平衡，这种“机械相互作用”控制着各种各样的细胞功能，包括细胞命运、形状和运动。尽管这一重要性，细胞的感觉和响应的细胞外基质的机械性质的分子机制还没有完全理解。我们的实验室证实，cAMP依赖性蛋白激酶（PKA）在细胞的前沿富集和激活，这种定位对细胞迁移很重要。在我们不断努力阐明PKA在细胞迁移过程中的空间调节机制的过程中，我们最近发现，PKA的局部活性受细胞张力的调节。具体地，在肌动球蛋白收缩性抑制后，前沿PKA活性迅速丧失。此外，当细胞被机械拉伸时，PKA在拉伸方向上以张力依赖性方式快速和局部激活。最后，PKA的抑制还阻断硬旋转-由ECM刚性和细胞-基质张力的梯度引导的细胞迁移。我们目前的重点是了解耦合细胞张力PKA的局部调节的机制。最近的初步数据表明，细胞收缩性和PKA的局部激活都依赖于细胞外Ca 2+通过牵张激活通道TRPM 7的流入。另外的数据有力地表明，G蛋白偶联受体-A2 B腺苷受体（AD 0 RA 2B）-在该机制中也起重要作用。基于我们的观察，我们假设，在迁移细胞的前沿PKA的局部激活是由机械化学机制，涉及局部细胞张力增加，细胞外Ca 2+的流入，和ADORA 2B的激活之间的相互作用。我们将通过确定ADORA 2B在细胞迁移过程中PKA机械活化中的作用，描述细胞迁移过程中ADORA 2B机械化学活化/调节的机制，并确定收缩性、Ca 2+和ADORA 2B在调节局部PKA活性和细胞迁移中的机制层次来检验这一假设。在拟议的研究结束时，我们将建立一个范例的途径，其中本地化耦合的细胞力学GPCR信号级联调节细胞骨架动力学和细胞运动。

 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.