Activation of G Protein-Coupled Kinase 2

描述（由申请人提供）：G蛋白偶联受体激酶2（GRK 2）不仅通过磷酸化活化的β-肾上腺素能受体，从而启动其脱敏，而且还通过隔离活化的G蛋白α和β-γ亚基来调节心脏中的异源三聚体G蛋白信号传导。尽管GRK 2在适应中具有有益的作用，但其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GRK 2和γ射线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，并且是第一次描述Gbetagamma绑定到真正的效应器靶点。该提案旨在解决GRK 2：Gbetagamma结构产生的一些问题，特别是与Gbetagamma、磷脂和GPCR激活GRK 2的机制有关的问题。 第一个目的是通过确定与磷脂或磷脂头基复合的GRK 2：G β γ的共晶结构，以及通过确定与核苷酸类似物和肽底物复合的GRK 2：G β γ的新结构来表征GRK 2的配体结合位点。我们还将通过将它们对接到激酶结构域来模拟对应于已知GRK 2磷酸化的肽，不仅可以了解更多关于GRK 2的序列特异性，而且还可以开发更好的肽底物或抑制剂。第二个目的是定义由G β γ结合引起的GRK 2的构象变化，主要是通过从现有晶体中确定GRK 2的胞质形式的结构。此外，将使用有限的蛋白水解和配体结合测定来评估配体或膜易位诱导的GRK 2的构象变化。第三个目的是确定GRK 2的受体对接位点。首先，将测试预测的对接位点内的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GRK 2：G β γ和活化的β 2-肾上腺素能受体之间的复合物的现有晶体的方法，最终目标是确定其晶体结构。在另一种方法中，将确定GRK 2：G β γ与对应于β 2-肾上腺素能受体的一个或多个胞质环的肽或与模拟GPCR的催化活性的肽mastoparan复合的结构。

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.