Molecular Electron Mircroscopy of Signaling Protein Complexes.

[摘要] G protein-coupled receptors (GPCRs) are the primary signal transduction units responsible for the communication between cells and their environment. Thus, GPCRs are involved in every aspect of human physiology and are currently the target of more than 30% of drugs.Understanding the activation, signaling, and silencing of GPCRs is crucial for designing therapeutic strategies and modern agents for treating several pathologies.Here we applied single particle electron microscopy (EM) analysis to obtain the first snapshots of agonist occupied beta-2-adrenergic receptor, a well-studied model GPCR, in complex with its primary signal transducer, the heterotrimeric G protein Gs (G-alpha-s/beta/gamma).EM 2D averages and 3D reconstructions of the detergent-solubilized complex revealed the complex architecture, and, unexpectedly, showed that the alpha-helical (AH) domain of G-alpha-s is highly flexible in the absence of nucleotide. In contrast, the presence of the pyrophosphate mimic foscarnet, and also the presence of GDP favor the stabilization of the AH domain on the Ras-like domain of G-alpha-s. Furthermore, we employed single particle EM to study the 3D architecture of beta-2-adrenergic receptor in complex with beta-arrestin-1, which is responsible for GPCR silencing and internalization. To this end, we took advantage of an optimized beta-2-adrenergic receptor/beta-arrestin-1 preparation that was based on complex stabilization by a Fab antibody. The 3D reconstructions and modeling of the beta-2-adrenergic receptor/beta-arrestin-1 complex provide novel insights into arrestin binding on GPCRs. Thus, herein we describe a starting framework to understand the structural basis of GPCR activation, signaling and regulation.Additionally, epigenetic regulation and signaling is a critical aspect of cellular function as well.We have reconstituted fully functional yeast and human Set1/COMPASS complex, the first H3K4 methylase, in vitro and have identified the minimum subunit composition required for histone H3K4 methylation.3D cryo-EM reconstructions of the core yeast complex, combined with immunolabeling and 2D EM analysis of the individual subcomplexes reveal a Y-shaped architecture with, the SET domain of Set1 is located at the juncture of Cps50, Cps30 and the Cps60-Cps25 module, lining the walls of a central channel that may act as the platform for catalysis and regulative processing of various degrees of H3K4 methylation.

[发布机构] University of Michigan

[效力级别] Signaling Protein Complexes

[关键词] Electron Microscopy;Signaling Protein Complexes;Biochemistry;Biological Chemistry;Science;Biological Chemistry