Progesterone Receptor and the hsp90 Chaperone Pathway

黄体酮受体与hsp90伴侣通路。当在胞浆提取物中处于失活状态时，禽类黄体酮受体（PR）与大多数类固醇受体和其他几种细胞信号蛋白一样，与热休克蛋白hsp90络合。这种复合物不是通过简单的结合形成的，而是通过一个需要ATP水解和一些额外蛋白质的过程形成的。参与受体复合体形成的其他蛋白有hsp70、三种hsp70共伴侣、hsp40、Hip和Hop、四种hsp90结合TPR蛋白和p23。我们已经开发了一种化学定义的系统，该系统具有纯化蛋白质的能力，可以组装带有hsp90的PR复合物。在体外组装过程中，只需要上述5种蛋白质，就可以通过3个步骤产生具有激素结合活性的PR。提出的研究的目的是进一步了解受体复合物的组装，它们在激素结合时的解离，以及受体相关蛋白的功能意义。这将通过广泛使用体外定义的组装系统来完成。PR复合物形成的每一步都将根据蛋白质相互作用的动力学和ATP的利用来表征。将对该系统进行修改，以便研究PR与hsp90的激素依赖性解离。这可能需要鉴定离解过程所需的新因素。最后，我们将描述作为结合hsp70和hsp90的相互作用位点的PR的结构域或元件，并研究促进热休克蛋白识别的特性。这些研究将导致对类固醇受体激活过程的机制描述，并为理解分子伴侣hsp70和hsp90的作用机制提供有价值的信息。

Progesterone receptor and the hsp90 chaperone pathway. When in its inactive form in cytosol extracts, the avian progesterone receptor (PR), like most steroid receptors and several other cell signaling proteins, is complexed with the heat shock protein hsp90. This complex does not form through a simple association, but through a process that requires ATP hydrolysis and several additional proteins. Other proteins involved in receptor complex formation are hsp70, three hsp70 co-chaperones, hsp40, Hip and Hop, four hsp90-binding TPR proteins and p23. We have developed a chemically defined system with purified proteins that has the capacity to assemble PR complexes with hsp90. Only five of the above proteins are needed for this in vitro assembly process which appears to proceed through three steps to generate PR with hormone binding activity. The objective of the proposed studies is to further our understanding on the assembly of receptor complexes, their dissociation upon hormone binding, and the functional significance of the receptor-associated proteins. This will be accomplished by extensive use of the in vitro defined assembly system. Events at each step of PR complex formation will be characterized in terms of the dynamics of protein interactions and the utilization of ATP. Modifications in the system will be made that allow the study of hormone- dependent dissociation of PR from hsp90. This will probably require the identification of new factors needed for the dissociation process. Finally, we will characterize the domains or elements of the PR that are interaction sites for binding hsp70 and hsp90 and investigate the properties which promote heat shock protein recognition. These studies should lead toward a mechanistic description of the steroid receptor activation process and should provide valuable information for understanding the mechanisms of action of the molecular chaperones hsp70 and hsp90.