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

G蛋白偶联受体激酶调节的分子基础

基本信息

批准号：
8281593
负责人：
John Tesmer
金额：
$ 41.07万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-15 至 2013-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8281593
关键词：
ADRBK1 gene Active Sites Adrenergic Receptor Affinity Binding Biochemical Biological Assay Biological Factors C-terminal Cardiovascular Diseases Cattle Cell physiology Cells Complex Crystallization Development Docking Drug Delivery Systems Engineering Enzymes Esthesia Family G Protein-Coupled Receptor Genes G Protein-Coupled Receptor Kinase Family G protein coupled receptor kinase G-Protein-Coupled Receptors GRK1 gene GRK6 gene GTP-Binding Proteins Health Heart Contractilities Heart failure Human Genome Hypertension Invertebrates Investigation Knowledge Lead Learning Length Libraries Light Membrane Molecular Molecular Conformation Mutagenesis N-terminal Nucleotides Opsin Peptides Pharmaceutical Preparations Phosphorylation Phosphotransferases Play Process Publishing RNA Regulation Relative (related person)Resolution Rhodopsin Role Signal Transduction Site Staging Structure Surface Tail Testing Therapeutic Agents Transducin Vertebrates Work X-Ray Crystallography aptamer base design human disease inhibitor/antagonist insight kinase inhibitor member novel novel strategies peptidomimetics receptor receptor coupling rhodopsin kinase tool

项目摘要

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.

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，控制从光感到心脏收缩力的过程。 GPCR 激酶 (GRK) 家族通过磷酸化细胞质环和 C 末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传导造成的损害，但异常的 GRK 活性与高血压和心力衰竭等人类疾病有关。 GRK 活性的抑制也有望增强许多针对 GPCR 的药物的作用。在过去五年中，我们的实验室在了解该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 GRK1（视紫红质激酶）、GRK2（2-肾上腺素能受体激酶 1）和 GRK6，以及与异三聚体 G1q 和 G23 亚基复合的 GRK2 结构。虽然我们对 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有了很多了解，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 GPCR 并被 GPCR 变构激活。在本提案的第一个目标中，我们测试了源自 GRK6 封闭构象的突破性结构的假设，其中保守的 N 末端螺旋与激酶结构域对接并将其稳定在更活跃的状态。该螺旋从激酶结构域延伸出来，因此它可以与 GPCR 相互作用，其方式类似于转导蛋白 C 端螺旋与视蛋白的结合方式。第二个目标致力于 GRK 受体复合物的晶体学分析。我们将研究 GRK6 与源自 GPCR 磷酸受体位点的底物肽复合物的闭合构象结构。为了帮助确定 GRK 如何与受体对接，我们将开发源自 GRK N 末端螺旋的肽和/或拟肽，它们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 的复合体的结构，我们将其设计为更容易呈现闭合构象。我们的最终目标是使用晶体学方法来定义新型 RNA 适体如何以高亲和力和选择性抑制 GRK2 的分子基础。我们将开发一种方法来筛选针对与适配体结合的 GRK2 表面关键口袋的选择性化合物，并将尝试设计对 GRK6 具有选择性的新适配体。了解 GPCR 如何激活 GRK 并表征这些酶上独特且功能关键的位点是开发可选择性调节细胞中 GRK 功能的试剂的关键。公共健康相关性：G 蛋白偶联受体 (GPCR) 激酶 (GRK) 磷酸化，从而调节人类基因组中约 800 个 GPCR 中大部分的活性。一些 GRK，例如 GRK2，与心血管疾病和高血压的进展密切相关。该提案研究了 GRK 如何识别其目标 GPCR 并受其调节的分子基础，并寻求从结构上表征一种新型 GRK 抑制剂，该抑制剂可能导致治疗药物或新分子工具的开发，以剖析细胞中的 GRK 功能。