STRUCTURE AND BIOLOGY OF BETA-ADRENERGIC RECEPTORS

我建议研究G蛋白连接受体(GPLR)的调节机制；GPLR C末端的结构决定因素，使其能够被酪氨酸激酶反向调节；以及参与信号传递的GPLR复合体的组织。该提案集中在哺乳动物β2肾上腺素能受体(Beta2AR)的C末端、细胞质的“尾巴”上，将其结构和功能作为一个离散的结构域进行分析。(1)我们将定义酪氨酸激酶(例如，胰岛素受体)通过哪些结构决定因素使β2AR磷酸化，并引发蛋白质-蛋白质与与胰岛素对β2AR的反向调节相关的接头分子和酶的相互作用。将使用与磷酸化平行的突变以及GFP-Beta2AR的共聚焦显微镜来测试反调节生长因子刺激Beta2AR的隔离的能力。(2)我们将分析Beta2AR与蛋白激酶、磷酸酶、酶、接头和支架蛋白(如AKAP 250所示)络合的大分子性质。将在激活、脱敏和隔离的条件下测试动态结合，利用蛋白质-蛋白质相互作用服从于共免疫沉淀和/或分离到固定的亲和基质。将使用几种互补的方法来测试蛋白质在与Beta2AR的复合体中的功能作用，包括通过反义技术消除目标蛋白质，将感兴趣的结构域或蛋白质片段表达为可能的抑制/激活剂，以及通过对目标蛋白质的特定结构域进行突变。(3)我们将在大肠杆菌中表达足够数量的C-末端尾巴，使其能够作为几种激酶和哺乳动物细胞蛋白质磷酸化的底物进行详细分析，以测试Beta2AR的这一区域(或特定片段)改变信号的能力。此外，我们还将合作进行大规模的C-末端结构域的分离、结晶和X射线分析。使用荧光共聚焦显微镜和针对复杂元件和/或GFP标记的融合蛋白的抗体进行的补充研究将补充和进一步测试生化和细胞生物学研究中关于C末端在逆调节生长因子调节Beta2AR中的作用的数据衍生。

I propose to study the mechanism of regulation of G-protein-linked receptors (GPLR); the structural determinants of the C-terminus of GPLR enabling counterregulation by tyrosine kinases; and the organization of GPLR complexes involved in signaling. The proposal focuses upon the C-terminal, cytoplasmic "tail" of the mammalian beta2-adrenergic receptor (beta2AR), approaching the analysis of its structure and function as a discrete domain. (1) We will define the structural determinants by which tyrosine kinases (e.g., insulin receptors) phosphorylate the beta2AR and provoke protein-protein interactions with adaptor molecules and enzymes relevant to counterregulation of the beta2AR by insulin. The ability of counterregulatory growth factors to stimulate sequestration of the beta2AR will be tested using mutagenesis in parallel with phosphorylation as well as confocal microscopy with the GFP-beta2AR. (2) We will analyze the macromolecular nature of beta2AR in complexation with protein kinases, phosphatases, enzymes, adaptor and scaffold proteins (as shown for AKAP 250). Dynamic association will be tested under conditions of activation, desensitization, and sequestration exploiting protein-protein interactions amenable to co-immuneprecipitation and/or isolation to immobilized affinity matrices. Functional roles for proteins in complex with beta2AR will be tested using several complementary approaches including elimination of target proteins by antisense technology, expression of fragments of the domain or protein of interest as possible inhibitors/activators, and via mutagenesis of specific domains of the target protein. (3) We will express the C-terminal tail in E.coli in sufficient quantities to allow its detailed analysis as a substrate for protein phosphorylation by several kinases and in mammalian cells to test the ability of this region (or specific fragments) of the beta2AR to alter signaling. In addition, we will collaborate on large-scale isolation, crystallization, and X-ray analysis of the C-terminal domain. Complementary studies performed with epifluorescence confocal microscopy and antibodies specific to complex elements and/or GFP-tagged fusion proteins of interest will complement and test further the data derivative of biochemical and cell biology studies on the role of the C-terminus in the regulation of beta2AR by counterregulatory growth factors.