G PROTEIN COUPLED RECEPTORS (GPCRS) G PROTEIN COMPLEX

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。Sprang和Sunahara已经产生了一种由His6序列连接到Galpas的Beta2肾上腺素能受体(Beta2AR)的融合蛋白。在Gbetagamma存在的情况下，这种融合蛋白能够与Beta2AR激动剂特异结合，并支持Galphas结构域的稳态GTP水解。β2AR-GalphasGbeta1Gamma2的复合体可以在昆虫细胞中从杆状病毒载体中表达，并纯化到毫克量的均一。用十二烷基麦芽糖苷进行膜萃取，可得到均一胶束，胶束中含有β2AR-GalphasGbeta1Gamma2。得到了~145 kDa的单体络合物和二聚体络合物。异质性的来源可能来自构象变化和可变的磷酸化。通过刺激cAMP的产生和钙离子的激活，β2AR调节心肌和骨骼肌、血管压力和葡萄糖代谢。总体而言，GPCRs是神经内分泌、免疫和感觉现象的基本调节器，因此是治疗广泛疾病的主要药物靶点。目前在分子水平上还不清楚gpr的作用机制。已经与NCMI的工作人员合作，对玻璃冰嵌入和负染的Beta2AR-GalphasGbeta1Gamma2粒子进行了单粒子成像。对负染颗粒图像的分析已经证明了获得不同类别平均值的可行性，从而能够计算三维重建。这些数据表明，β2AR-GalphasGbeta1 Gamma2粒子大小均匀，形状不对称，但存在异质性。异质性的来源正在调查中，针对天然络合物的单抗正在被开发作为佐剂，以提高络合物的稳定性，增加络合物的大小和不对称性，以改进EM分析。我们试图用冷冻EM单粒子成像和X射线结晶学来获得β2AR-GalphasGbeta1 Gamma2络合物的三维结构，在功能相关的构象状态下。这项努力是一个长期研究计划的一部分，目的是阐明G蛋白信号转导的机制。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Sprang and Sunahara have produced a fusion protein of beta2 adrenergic receptor (beta2AR) linked by a his6 sequence, to Galphas. This fusion protein, in the presence of Gbetagamma is able to bind specifically to beta2AR agonists and supports steady state GTP hydrolysis by the Galphas domain. The complex of beta2AR-GalphasGbeta1gamma2 can be expressed from baculovirus vectors in insect cells and purified to homogeneity in milligram quantities. Membrane extraction with dodecyl maltoside yields homogeneous micelles containing beta2AR-GalphasGbeta1gamma2. Both monomeric (~145 kDa) and dimeric complexes have been obtained. Sources of heterogeneity may derive from conformational variation and variable phosphorylation. By stimulating cAMP production and activation of Ca2+, beta2AR regulates cardiac and skeletal muscle, vascular pressure and glucose metabolism. GPCRs in general are essential regulators of neuro-endocrine, immune and sensory phenomena and are consequently major drug targets for a broad range of disorders. The mechanism of GPCR function is not understood at the molecular level. Single particle imaging of vitreous ice-embedded and negative stained beta2AR-GalphasGbeta1gamma2 particles have been undertaken in collaboration with NCMI staff. Analysis of images of negatively stained particles has demonstrated the feasibility of obtaining distinct class averages, enabling calculation of a 3-D reconstruction. These data demonstrate that beta2AR-GalphasGbeta1gamma2 particles are uniform in size, asymmetric in shape, but suffer from heterogeneity. The source of heterogeneity is under investigation, and monoclonal antibodies raised against native complexes are being developed as adjuvants to improve the stability and increase the size and asymmetry of the complexes for improved EM analysis. We seek to obtain the 3-D structure of the beta2AR-GalphasGbeta1gamma2 complex using both cryo-EM single particle imaging and x-ray crystallography, in functionally relevant conformational states. This effort is part of a long-standing research program to elucidate the mechanism of G-protein signaling.