Molecular basis for the regulation of G protein-coupled receptor kinases

描述(申请人提供)：G蛋白偶联受体(GPCRs)是细胞生理学的关键调节器，控制从光的感觉到心脏的收缩的各种过程。一个GPCRK家族通过磷酸化胞质环和C末端的位点来调节这些GPCRs的活性。尽管GRK可以使细胞适应并保护它们免受持续信号的损害，但GRK活性的异常与高血压和心力衰竭等人类疾病有关。抑制GRK活性也有望增强许多靶向GPCRs的药物的作用。在过去的五年里，我们的实验室在了解这个激酶家族的结构和功能方面取得了重大进展。我们已经获得了代表所有三个GRK亚家族的高分辨晶体结构，包括GRK1(视紫红质激酶)、GRK2(2-肾上腺素能受体激酶1)和GRK6，以及GRK2与异三聚体G1q和G23亚基的复合体结构。虽然关于GRK的模块结构、它们与G蛋白的相互作用以及它们在膜上的配置已经了解了很多，但直到最近我们才确定了一种晶体结构，这使得我们能够合理地测试GRK是如何识别GPCRs并被GPCRs变构激活的。在这一提议的第一个目标中，我们在封闭的构象中测试来自我们突破的GRK6结构的假说，其中保守的N-末端螺旋与激活域对接，并将其稳定在更活跃的状态。该螺旋从激活域延伸而来，因此它可以以类似于转导蛋白的C末端螺旋结合视蛋白的方式与GPCR相互作用。第二个目标是GRK受体复合体的结晶学分析。我们将在GRK6与来自GPCRs的磷受体位点的底物多肽的复合体中寻找闭合构象的结构。为了帮助确定GRKs是如何对接在受体上的，我们将开发来自GRKs N-末端螺旋的多肽和/或多肽模拟物，它们与激活的牛或头足类视紫红质高亲和力结合，用于共结晶筛选。我们还将尝试确定这些原型GPCR的结构与全长GRK的复合体，我们设计的GRK更容易假设闭合构象。我们的最终目标是用结晶学的方法来定义一个新的RNA适配子如何高亲和力和选择性地抑制GRK2的分子基础。我们将开发一种方法来筛选靶向适体结合的GRK2表面关键口袋的选择性化合物，并将尝试设计对GRK6具有选择性的新适体。了解GPCRs如何激活GRK并确定这些酶上独特的和功能关键的位点是开发能够选择性调节细胞中GRK功能的试剂的关键。与公共健康相关：G蛋白偶联受体(GPCRK)使磷酸化，从而调节人类基因组中约800个GPCRs中的大多数的活性。一些GRK，如GRK2，与心血管疾病和高血压的进展密切相关。这项建议研究了GRK如何识别其靶GPCRs并受其调控的分子基础，并试图从结构上表征一种新的GRK抑制剂，该抑制剂可能导致治疗药物的开发或新的分子工具来剖析细胞中的GRK功能。

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) are key regulators of cell physiology, controlling processes that range from the sensation of light to the contractility of the heart. A family of GPCR kinases (GRKs) modulates the activity of these GPCRs by phosphorylating sites in their cytoplasmic loops and C-terminal tails. Although GRKs allow cells to adapt and can protect them from damage incurred by sustained signaling, aberrant GRK activity has been associated with human disease such as hypertension and heart failure. Inhibition of GRK activity is also expected to enhance the action of the many drugs that target GPCRs. In the last five years, our lab has made significant progress in understanding the structure and function of this kinase family. We have produced high resolution crystal structures that represent all three GRK subfamilies, including that of GRK1 (rhodopsin kinase), GRK2 (2-adrenergic receptor kinase 1), and GRK6, as well as structures of GRK2 in complex with heterotrimeric G1q and G23 subunits. While much has been learned about the modular structure of GRKs, their interactions with G proteins, and their configuration at the membrane, only recently have we determined a crystal structure that permits us to rationally test how GRKs recognize and are allosterically activated by GPCRs. In the first aim of this proposal, we test hypotheses derived from our breakthrough structure of GRK6 in a closed conformation, wherein a conserved N-terminal helix docks with the kinase domain and stabilizes it in a more active state. This helix extends from the kinase domain such that it could interact with a GPCR in a manner analogous to how the C-terminal helix of transducin binds opsin. The second aim is devoted to crystallographic analysis of GRK-receptor complexes. We will pursue structures of the closed conformation of GRK6 in complex with substrate peptides derived from the phosphoacceptor sites of GPCRs. To help define how GRKs dock on the receptor, we will develop peptides and/or peptidomimetics derived from the N-terminal helices of GRKs that bind with high affinity to activated bovine or cephalopod rhodopsin for co-crystallization screens. We will also attempt to determine structures of these prototypical GPCRs in complex with full-length GRKs that we engineer to more readily assume a closed conformation. Our final aim is to use a crystallographic approach to define the molecular basis for how a novel RNA aptamer inhibits GRK2 with high affinity and selectivity. We will develop an assay to screen for selective compounds that target key pockets on the surface of GRK2 bound by the aptamer, and will attempt to engineer new aptamers that are selective for GRK6. Understanding how GPCRs activate GRKs and characterizing the unique and functionally critical sites on these enzymes is key to the development of agents that can selectively regulate GRK function in cells. PUBLIC HEALTH RELEVANCE: G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate and thereby regulate the activity of most of the ~800 GPCRs in the human genome. Some GRKs, such as GRK2, are strongly implicated in the progression of cardiovascular disease and hypertension. This proposal investigates the molecular basis for how GRKs recognize and are regulated by their target GPCRs, and seeks to structurally characterize a novel GRK inhibitor that could lead to the development of therapeutic agents or new molecular tools to dissect GRK function in cells.