Dataset Information

Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors.

ABSTRACT: Structural studies of human G protein-coupled receptors (GPCRs) have recently been accelerated through the use of a fusion partner that was inserted into the third intracellular loop. Using chimeras of the human ?(2)-adrenergic and human A(2A) adenosine receptors, we present the methodology and data for the initial selection of an expanded set of fusion partners for crystallizing GPCRs. In particular, use of the thermostabilized apocytochrome b(562)RIL as a fusion partner displays certain advantages over previously utilized fusion proteins, resulting in a significant improvement in stability and structure of GPCR-fusion constructs.

SUBMITTER: Chun E

PROVIDER: S-EPMC3375611 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors.

Chun Eugene E Thompson Aaron A AA Liu Wei W Roth Christopher B CB Griffith Mark T MT Katritch Vsevolod V Kunken Joshua J Xu Fei F Cherezov Vadim V Hanson Michael A MA Stevens Raymond C RC

Structure (London, England : 1993) 20120601 6

Structural studies of human G protein-coupled receptors (GPCRs) have recently been accelerated through the use of a fusion partner that was inserted into the third intracellular loop. Using chimeras of the human β(2)-adrenergic and human A(2A) adenosine receptors, we present the methodology and data for the initial selection of an expanded set of fusion partners for crystallizing GPCRs. In particular, use of the thermostabilized apocytochrome b(562)RIL as a fusion partner displays certain advant ...[more]

PMID: 22681902

Similar Datasets

Project description:Understanding how neuromodulators regulate behavior requires investigating their effects on functional neural systems, but also their underlying cellular mechanisms. Utilizing extensively characterized lamprey motor circuits, and the unique access to reticulospinal presynaptic terminals in the intact spinal cord that initiate these behaviors, we investigated effects of presynaptic G-protein-coupled receptors on locomotion from the systems level, to the molecular control of vesicle fusion. 5-HT inhibits neurotransmitter release via a Gbetagamma interaction with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that promotes kiss-and-run vesicle fusion. In the lamprey spinal cord, we demonstrate that, although presynaptic 5-HT receptors inhibit evoked neurotransmitter release from reticulospinal command neurons, their activation does not abolish locomotion but rather modulates locomotor rhythms. Liberation of presynaptic Gbetagamma causes substantial inhibition of AMPA receptor-mediated synaptic responses but leaves NMDA receptor-mediated components of neurotransmission mostly intact. Because Gbetagamma binding to the SNARE complex is displaced by Ca(2+)-synaptotagmin binding, 5-HT-mediated inhibition displays Ca(2+) sensitivity. We show that, as Ca(2+) accumulates presynaptically during physiological bouts of activity, 5-HT/Gbetagamma-mediated presynaptic inhibition is relieved, leading to a frequency-dependent increase in synaptic concentrations of glutamate. This frequency-dependent phenomenon mirrors a shift in the vesicle fusion mode and a recovery of AMPA receptor-mediated EPSCs from inhibition without a modification of NMDA receptor EPSCs. We conclude that activation of presynaptic 5-HT G-protein-coupled receptors state-dependently alters vesicle fusion properties to shift the weight of NMDA versus AMPA receptor-mediated responses at excitatory synapses. We have therefore identified a novel mechanism in which modification of vesicle fusion modes may profoundly alter locomotor behavior.

Project description:The years 2000 and 2007 witnessed milestones in current understanding of G protein-coupled receptor (GPCR) structural biology. In 2000 the first GPCR, bovine rhodopsin, was crystallized and the structure was solved, while in 2007 the structure of ?(2)-adrenergic receptor, the first GPCR with diffusible ligands, was determined owing to advances in microcrystallization and an insertion of the fast-folding lysozyme into the receptor. In parallel with those crystallographic studies, the biological and biochemical characterization of GPCRs has advanced considerably because those receptors are molecular targets for many of currently used drugs. Therefore, the mechanisms of activation and signal transduction to the cell interior deduced from known GPCRs structures are of the highest importance for drug discovery. These proteins are the most diversified membrane receptors encoded by hundreds of genes in our genome. They participate in processes responsible for vision, smell, taste and neuronal transmission in response to photons or binding of ions, hormones, peptides, chemokines and other factors. Although the GPCRs share a common seven-transmembrane ?-helical bundle structure their binding sites can accommodate thousands of different ligands. The ligands, including agonists, antagonists or inverse agonists change the structure of the receptor. With bound agonists they can form a complex with a suitable G protein, be phosphorylated by kinases or bind arrestin. The discovered signaling cascades invoked by arrestin independently of G proteins makes the GPCR activating scheme more complex such that a ligand acting as an antagonist for G protein signaling can also act as an agonist in arrestin-dependent signaling. Additionally, the existence of multiple ligand-dependent partial activation states as well as dimerization of GPCRs result in a 'microprocessor-like' action of these receptors rather than an 'on-off' switch as was commonly believed only a decade ago.

Dataset Information

Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors.

Publications

Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets