Molecular basis for arrestin-mediated signaling

项目摘要 Arrestin蛋白是G蛋白偶联受体（GPCR）信号传导的主要调节剂，并且以两种方式起作用。 首先，抑制蛋白通过物理阻断G蛋白来终止G蛋白与同源受体的偶联 偶联位点第二，抑制蛋白可以支持G蛋白非依赖性信号传导。最好的研究逮捕- 介导的信号传导途径包括促分裂原活化蛋白（MAP）激酶的活化。最近我们 已经确定了激活的arrestin-3的结构，其显示了预期的域间扭曲。我们也 确定的局部，以前未识别的构象变化的效应结合区的 激活抑制蛋白结合结构分析和功能测量，我们提出了一个新的范式 对于抑制蛋白支架效应器通过这些网站，其中抑制蛋白的过渡到活性构象 可以“开启”效应子结合。在功能上类似于G蛋白的“开关区”，这些激活- 效应物结合区的依赖性构象变化距离受体位点30-40 μ m（或 非受体激活剂）结合，并且与先前的激活相关构象变化不同 在文献中描述。我们利用这一新发现，有三个目标，探索之间的联系， 激活和效应子结合三个目标。 在目标1中，我们结合联合收割机体外结构和生物物理技术（X射线晶体学，多角度激光 光散射（MALLS）和亲和性测量，用双电子电子共振（DEER）作为 另一种方法），在细胞中进行单分子和功能测量，以研究 寡聚化在稳定受体非依赖性抑制蛋白激活中的作用。 在目标2中，我们将联合收割机诱变和功能分析相结合，以揭示磷酸抑制蛋白和抑制蛋白是如何被激活的。 在抑制蛋白激活位点的蛋白质相互作用被变构传递到效应物结合位点， 其在30 - 40 mm远侧。 在目标3中，我们使用新的方法来稳定arrestin-3的活性形式，并使用X射线晶体学来 识别抑制蛋白-效应物相互作用的细节。

PROJECT SUMMARY Arrestin proteins are master regulators of G protein coupled receptor (GPCR) signaling, and act in two ways. First, arrestins terminate the coupling of G proteins to cognate receptor by physically blocking the G protein coupling site. Second, arrestins can support G protein independent signaling. The best studied arrestin- mediated signaling pathways include the activation of mitogen activated protein (MAP) kinases. Recently we have determined the structure of activated arrestin-3, which shows the expected inter-domain twist. We also identified localized, previously unrecognized conformational changes in the effector binding regions of activated arrestin. Combining structural analysis with functional measurements, we propose a new paradigm for arrestin scaffolding of effectors via these sites, where the transition of arrestin into the active conformation can “turn on” effector binding. Functionally analogous to `switch regions' of G proteins, these activation- dependent conformational changes in effector binding regions are 30-40 Å distal from the site of receptor (or non-receptor activator) binding and are distinct from activation-associated conformational changes previously described in the literature. We leverage this new finding with three aims that explore the connection between activation and effector binding in three aims. In Aim 1, we combine in vitro structural and biophysical techniques (X-ray crystallography, multi-angle laser light scattering (MALLS), and affinity measurements, with double electron electron resonance (DEER) as an alternative approach) with single molecule and functional measurements in cells to investigate the role of oligomerization in stabilizing receptor-independent arrestin activation. In Aim 2, we combine mutagenesis and functional analysis to reveal how arrestin-phosphate and arrestin- protein interactions at the activation sites of arrestin is allosterically transmitted to the effector binding sites, which are 30 – 40 Å distal. In Aim 3, we use new methods to stabilize the active form of arrestin-3 and use X-ray crystallography to identify the details of arrestin-effector interactions.