Activation of G Protein-Coupled Kinase 2

G 蛋白偶联激酶 2 的激活

• 批准号: 6837604
• 负责人: John Tesmer
• 金额: $ 10.18万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6837604
• 关键词: G protein; G protein coupled receptor kinase; SDS polyacrylamide gel electrophoresis; autoradiography; beta adrenergic receptor; binding sites; biological signal transduction; cell line; cell membrane; enzyme activity; ligands; phosphorylation; protein structure function; radioimmunoassay; site directed mutagenesis

DESCRIPTION (provided by applicant): G protein-coupled receptor kinase 2 (GRK2) regulates heterotrimeric G protein signaling in the heart not only by phosphorylating activated beta-adrenergic receptors, thereby initiating their desensitization, but also by sequestering activated G protein alpha and betagamma subunits. Despite its beneficial role in adaptation, unusually high expression of GRK2 is strongly implicated in the onset of cardiovascular disease. We recently determined the crystal structure of a peripheral membrane complex between GRK2 and Gbetagamma. The structure reveals the core architecture shared by all GRKs, and is the first description of Gbetagamma bound to a bona fide effector target. This proposal seeks to address some of the questions generated by the GRK2:Gbetagamma structure, particularly those pertaining to the mechanism of activation of GRK2 by Gbetagamma, phospholipids and GPCRs. The first aim is to characterize the ligand binding sites of GRK2 by determining co-crystal structures of GRK2:Gbetagamma in complex with phospholipids or phospholipid head groups, and by determining novel structures of GRK2:Gbetagamma in complex with nucleotide analogs and peptide substrates. We will also model peptides corresponding to known GRK2 phosphorylation by docking them to the kinase domain, not only to learn more about the sequence specificity of GRK2, but also potentially to develop better peptide substrates or inhibitors. The second aim is to define the conformational changes induced in GRK2 by the binding of Gbetagamma, primarily by determining the structure of the cytosolic form of GRK2 from existing crystals. In addition, conformational changes in GRK2 induced by ligands or membrane translocation will be evaluated using limited proteolysis and ligand-binding assays. The third aim is to define the receptor-docking site of GRK2. First, site-directed mutants of various residues within the predicted docking site will be tested for their ability to block receptor phosphorylation. Secondly, methods to improve existing crystals of the complex between GRK2:Gbetagamma and the activated beta2-adrenergic receptor will be developed, with the ultimate goal of determining its crystallographic structure. In an alternative approach, structures will be determined of GRK2:Gbetagamma in complex either with peptides that correspond to one or more cytosolic loops of the beta2-adrenergic receptor or with a peptide, mastoparan, that mimics the catalytic activity of GPCRs.

描述（由申请人提供）：G蛋白偶联受体激酶2（GRK2）不仅通过磷酸化激活的β-肾上腺素能受体从而启动其脱敏，而且还通过隔离激活的G蛋白α和βγ来调节心脏中的异三聚体G蛋白信号传导亚单位。尽管 GRK2 在适应中发挥有益作用，但异常高的表达与心血管疾病的发病密切相关。我们最近确定了 GRK2 和 Gbetagamma 之间的外周膜复合物的晶体结构。该结构揭示了所有 GRK 共享的核心架构，并且是对与真正的效应器目标结合的 Gbetagamma 的第一个描述。该提案旨在解决 GRK2:Gbetagamma 结构产生的一些问题，特别是与 Gbetagamma、磷脂和 GPCR 激活 GRK2 的机制有关的问题。 第一个目标是通过确定 GRK2:Gbetagamma 与磷脂或磷脂头基复合物的共晶结构，以及确定 GRK2:Gbetagamma 与核苷酸类似物和肽底物复合物的新结构来表征 GRK2 的配体结合位点。我们还将通过将已知 GRK2 磷酸化对应的肽与激酶结构域对接来对其进行建模，不仅可以了解有关 GRK2 序列特异性的更多信息，而且有可能开发更好的肽底物或抑制剂。第二个目标是通过 Gbetagamma 的结合来定义 GRK2 中诱导的构象变化，主要是通过从现有晶体中确定 GRK2 胞质形式的结构。此外，配体或膜易位诱导的 GRK2 构象变化将使用有限的蛋白水解和配体结合测定进行评估。第三个目标是确定 GRK2 的受体对接位点。首先，将测试预测对接位点内各种残基的定点突变体阻断受体磷酸化的能力。其次，将开发改进GRK2:Gbetagamma与激活的β2-肾上腺素受体复合物现有晶体的方法，最终目标是确定其晶体结构。在另一种方法中，将确定GRK2:Gbetagamma与对应于β2-肾上腺素能受体的一个或多个胞质环的肽或与模拟GPCR催化活性的肽mastoparan复合物的结构。