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

描述（由申请人提供）：G蛋白偶联受体（gpcr）是细胞生理学的关键调节因子，控制从光的感觉到心脏收缩的过程。GPCR激酶家族（GRKs）通过磷酸化细胞质环和c端尾部的位点来调节这些GPCR的活性。尽管GRK允许细胞适应并保护它们免受持续信号引起的损伤，但异常的GRK活性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gpcr的药物的作用。在过去的五年中，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。我们已经制作了高分辨率的晶体结构，代表所有三个GRK亚家族，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亚基复合物的结构。虽然我们对grk的模块化结构、它们与G蛋白的相互作用以及它们在膜上的结构已经了解了很多，但直到最近我们才确定了一种晶体结构，使我们能够合理地测试grk如何识别并被gpcr变构激活。在本提案的第一个目标中，我们测试了基于封闭构象GRK6的突破性结构的假设，其中保守的n端螺旋与激酶结构域对接并将其稳定在更活跃的状态。这个螺旋从激酶结构域延伸出来，这样它就可以以类似于转导蛋白的c端螺旋结合视蛋白的方式与GPCR相互作用。第二个目标是致力于grk受体复合物的晶体学分析。我们将研究GRK6与来自gpcr磷酸化受体位点的底物肽复合物的封闭构象结构。为了帮助确定GRKs如何与受体对接，我们将开发从GRKs的n端螺旋衍生的肽和/或拟肽物，这些肽和/或拟肽物与活化的牛或头足类视紫红质的高亲和力结合，用于共结晶筛选。我们还将尝试确定这些原型gpcr与全长grk复合物的结构，我们设计的是更容易假设一个封闭的构象。我们的最终目标是使用晶体学方法来定义一种新型RNA适体如何以高亲和力和选择性抑制GRK2的分子基础。我们将开发一种检测方法来筛选靶向GRK2表面上由适配体结合的关键口袋的选择性化合物，并将尝试设计出对GRK6具有选择性的新适配体。了解gpcr如何激活GRK，并描述这些酶上独特和功能关键的位点，是开发能够选择性调节细胞中GRK功能的药物的关键。公共卫生相关性：G蛋白偶联受体（GPCR）激酶（GRKs）磷酸化，从而调节人类基因组中约800个GPCR中的大多数的活性。一些GRKs，如GRK2，与心血管疾病和高血压的进展密切相关。本研究旨在研究GRK如何识别并受其靶gpcr调控的分子基础，并寻求一种新型GRK抑制剂的结构特征，这可能会导致治疗剂或新的分子工具的发展，以解剖细胞中的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.