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性的过程。GPCR激酶（GRKs）家族通过使这些GPCR的胞质环和C末端尾中的位点磷酸化来调节这些GPCR的活性。尽管GRK允许细胞适应并可以保护它们免受持续信号传导引起的损伤，但异常的GRK活性与人类疾病如高血压和心力衰竭有关。GRK活性的抑制也有望增强靶向GPCR的许多药物的作用。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。我们已经产生了代表所有三个GRK亚家族的高分辨率晶体结构，包括GRK 1（视紫红质激酶），GRK 2（2-肾上腺素能受体激酶1）和GRK 6，以及GRK 2与异源三聚体G1 q和G23亚基复合的结构。虽然已经了解了很多关于GRKs的模块化结构，它们与G蛋白的相互作用，以及它们在膜上的构型，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GRKs如何识别GPCR并被GPCR变构激活。在这个建议的第一个目标，我们测试的假设来自我们的突破性结构GRK 6在一个封闭的构象，其中保守的N-末端螺旋码头与激酶结构域和稳定它在一个更活跃的状态。该螺旋从激酶结构域延伸，使得它可以以类似于转导蛋白的C末端螺旋如何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。我们将继续研究GRK 6与来自GPCR磷酸受体位点的底物肽复合的闭合构象的结构。为了帮助定义GRKs如何对接在受体上，我们将开发肽和/或肽模拟物，这些肽和/或肽模拟物衍生自GRKs的N-末端螺旋，其以高亲和力与活化的牛或头足类动物视紫红质结合，用于共结晶筛选。我们还将尝试确定这些原型GPCR与全长GRK的复合结构，我们设计这些GRK以更容易地呈现闭合构象。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GRK 2的分子基础。我们将开发一种检测方法来筛选选择性化合物，这些化合物靶向适体结合的GRK 2表面上的关键口袋，并将尝试设计对GRK 6具有选择性的新适体。了解GPCR如何激活GRK并表征这些酶上的独特和功能关键位点是开发可以选择性调节细胞中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种GPCR中大多数的活性。一些GRK，如GRK 2，与心血管疾病和高血压的进展密切相关。该提案研究了GRK如何识别并受其靶GPCR调控的分子基础，并试图在结构上表征一种新型GRK抑制剂，该抑制剂可能导致开发治疗剂或新的分子工具来剖析细胞中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.