Structural basis for reciprocal regulation of the GTPases tubulin and Gsalpha

描述(由申请人提供)： 某些Galpha蛋白是GPCR信号通路中的GTPase成分，可以与细胞骨架的一种成分微管蛋白形成复合体。值得注意的是，微管蛋白似乎反式激活Galpha(将GTP从微管蛋白直接转移到Galpha，反过来，Gs-α与微管加端结合激活微管蛋白GTP酶并增加微管动力学。微管蛋白和Gs-α之间的持续联系似乎将Gs-α从积极参与细胞信号传递中隔离出来。长期使用抗抑郁药物治疗细胞会将Gs-α转移到一个膜室，在那里它更多地与腺苷环化酶联系在一起，而与微管蛋白联系较少。一些滥用药物的长期治疗也可能改变膜信号隔间以及Gs-α和微管蛋白之间的关系。我们一直在试图了解这些鲜为人知的现象的分子基础。模拟研究表明，Gs-α中对GTP结合敏感的Switch1区域直接与微管蛋白的GTP结合位点相互作用；Galpha中阻止微管蛋白反式激活Galpha的突变也映射到这一区域。为了更深入地了解Galpha蛋白和微管蛋白之间调节复合体形成的相互作用，我们建议更全面地表征纯化的Gs-α/微管蛋白二聚体复合体，并确定不同核苷酸结合状态下异三聚体复合体的晶体结构。这些结构研究将提供对GTP酶调节的显著机制的直接洞察-在蛋白质结构和细胞骨架水平上-但重要的是，它们也将为随后针对Gs-α/微管蛋白界面的小分子的开发提供基础，这些小分子可能被证明对抑郁症和药物成瘾或依赖的治疗有用。这项工作代表着我们的实验室之间的协同融合，连接了生化、细胞生物学和结构生物学方法。它直接解决了一个重要的和新的调节相互作用的假说，这可能最终有助于从根本上理解与慢性药物治疗有关的分子和细胞事件，以及情绪和依赖的生物学。

DESCRIPTION (provided by applicant): Certain Galpha proteins, the GTPase component of the GPCR signaling pathways, can form complexes with tubulin, a component of the cytoskeleton. Remarkably, tubulin appears to transactivate Galpha (transferring GTP directly from tubulin to Galpha, and, inversly, Gs-alpha binding to microtubule plus-ends activates the tubulin GTPase and increases microtubule dynamics. Continued association between tubulin and Gs-alpha appears to sequester Gs-alpha from active involvement in cellular signaling. Chronic treatment of cells with antidepressant drugs moves Gs-alpha into a membrane compartment where it is more associated with adenylyl cyclase and less associated with tubulin. Chronic treatment with some drugs of abuse may also alter membrane signaling compartments and the relationship between Gs-alpha and microtubule proteins. we have been seeking to understand the molecular basis for these poorly understood phenomena. Modeling studies suggest that the switch1 regions of Gs-alpha that are sensitive to GTP binding interact directly with the GTP-binding site of tubulin; mutations in Galpha that block transactivation of Galpha by tubulin map to this region as well. In order to obtain deeper structural insight into the interactions that regulate complex formation between Galpha proteins and tubulin, we propose here to more fully characterize the purified Gs-alpha/tubulin dimer complex and to determine the crystal structure of the heterotrimeric complex in different nucleotide-bound states. These structural studies will provide direct insight into a remarkable mechanism of GTPase regulation - mediated at both the protein-structural and cytoskeletal levels - but importantly, they will also provide the basis for subsequent development of small molecules targeted to the Gs-alpha/tubulin interface that may prove useful in therapy for depression and drug addiction or dependency. The work represents a synergistic fusion of our laboratories bridging biochemical, cell biological and structural biological approaches. It addresses directly a significant and novel hypothesis for a regulatory interaction that that may ultimately contribute to fundamental understanding of the molecular and cellular events involved in response to chronic drug treatment and of the biology of mood and dependency.