Activation of G Protein-Coupled Kinase 2

描述(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它们的脱敏，而且通过隔离激活的G蛋白α和β亚基来调节心脏中的异三聚体G蛋白信号。尽管GRK2在适应中发挥了有益的作用，但GRK2的异常高表达与心血管疾病的发生密切相关。我们最近确定了GRK2和Gbetagamma之间的外周膜复合体的晶体结构。该结构揭示了所有GRK共享的核心架构，并且是对Gbetagamma绑定到真正的效应器目标的第一次描述。本提案旨在解决GRK2：Gbetagamma结构产生的一些问题，特别是与Gbetagamma、磷脂和GPCRs激活GRK2的机制有关的问题。 第一个目的是通过确定GRK2：Gbetagamma在与磷脂或磷脂头部基团的络合物中的共晶结构，以及通过确定GRK2：Gbetagamma在与核苷酸类似物和肽底物的络合物中的新结构来表征GRK2的配体结合部位。我们还将通过将已知的GRK2磷酸化对应的多肽对接到激活域来对其进行建模，不仅是为了更多地了解GRK2的序列特异性，而且还可能开发更好的多肽底物或抑制剂。第二个目的是确定GRK2中由Gbetagamma结合引起的构象变化，主要是通过从现有晶体中确定GRK2的胞浆形式的结构。此外，将使用有限的蛋白分解和配体结合分析来评估由配体或膜移位引起的GRK2的构象变化。第三个目的是确定GRK2的受体对接位置。首先，在预测的对接位置内的各种残基的定点突变体将被测试它们阻止受体磷酸化的能力。其次，将开发改进GRK2：Gbetagamma与激活的β2-肾上腺素能受体之间的络合物的现有晶体的方法，最终目的是确定其晶体结构。在另一种方法中，将确定GRK2：Gbetagamma的结构，要么与与β2-肾上腺素能受体的一个或多个胞浆环相对应的多肽，要么与模拟GPCRs催化活性的多肽Mastoparan。

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.