Structural basis for reciprocal regulation of the GTPases tubulin and Gsalpha

描述（由申请人提供）： 某些G α蛋白，GPCR信号通路的GT3组分，可以与微管蛋白（细胞骨架的组分）形成复合物。值得注意的是，微管蛋白似乎反式激活G α（将GTP直接从微管蛋白转移到G α，反之，与微管正末端结合的G α激活微管蛋白GTP酶并增加微管动力学。微管蛋白和Gs-alpha之间的持续关联似乎使Gs-alpha与细胞信号传导的积极参与隔离。 用抗抑郁药物对细胞进行慢性治疗将Gs-alpha移动到膜室中，在那里它与腺苷酸环化酶的相关性更高，与微管蛋白的相关性更低。一些滥用药物的慢性治疗也可能改变膜信号室以及Gs-alpha和微管蛋白之间的关系。我们一直在努力了解这些鲜为人知的现象的分子基础。建模研究表明，开关1区域的Gs-alpha是敏感的GTP结合直接相互作用的GTP结合位点的微管蛋白;突变的Galalpha阻止Galalpha的转录激活微管映射到这个区域以及。为了获得更深层次的结构洞察的相互作用，调节复合物的形成之间的Ga蛋白和微管蛋白，我们建议在这里更充分地表征纯化的GS-α/微管蛋白二聚体复合物，并确定在不同的核苷酸结合状态的异源三聚体复合物的晶体结构。这些结构研究将提供直接洞察到一个显着的机制GT3调节-介导的蛋白质结构和细胞骨架水平-但重要的是，他们也将提供基础，为后续开发的小分子靶向的Gs-alpha/微管蛋白接口，可能证明是有用的治疗抑郁症和药物成瘾或依赖。这项工作代表了我们实验室桥接生物化学，细胞生物学和结构生物学方法的协同融合。它直接解决了一个重要的和新的假说的监管相互作用，这可能最终有助于基本了解的分子和细胞的事件参与慢性药物治疗和生物学的情绪和依赖性。

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.