GPCR Membrane Preparations
Creative Biolabs' membrane preparations are useful for membrane protein research. We offer membrane preparations to study the role of membrane proteins in diseases. Membrane preparations from Creative Biolabs are quality-assured frozen membranes from cells expressing recombinant or natural receptors.
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Background of GPCR
G-protein-coupled receptors (GPCRs) mediate the majority of our physiological reactions to hormones, neurotransmitters, and environmental stimulants, and hence offer a wide range of therapeutic possibilities. All GPCRs are characterized by the presence of seven membrane-spanning -helical segments separated by alternating intracellular and extracellular loop sections at the most basic level. GPCRs have a unique set of signaling activities encompassing various G-protein subtypes, as well as G-protein-independent signaling pathways and intricate regulation processes.
Fig.1 Signal
transduction in G-protein-coupled receptors. (Rosenbaum, 2009)
Application of GPCR Membrane Preparations
Drug development has been transformed by the introduction of membrane ligand binding assays and high-throughput screening. Ligand binding assays allow us to see how a ligand or a new synthetic molecule interacts with a target protein and assess affinity and kinetics of those interactions, while HTS helps to automate research and shorten development time. Cell signaling and ligand binding in GPCRs are controlled by changes in the cell membrane and the membrane itself.
Published Data
| Paper Title | Conformational biosensors reveal adrenoceptor signalling from endosomes |
| Journal | Nature |
| Published | 2013 |
| Abstract | Canonical signal transduction, which is mediated by G-protein-coupled receptors (GPCRs) coupling to heterotrimeric G proteins, is long believed to be restricted to the plasma membrane. Analytical techniques with little to no subcellular resolution are the foundation of the evidence used to support this conventional viewpoint. Later, it was suggested that internalized GPCRs can indeed contribute to the acute G-protein-mediated response, or that internalized GPCR signaling is limited to G-protein-independent mechanisms like scaffolding by arrestins. It was also suggested that GPCR activation elicits a distinct type of persistent G protein signaling. The various latter hypotheses are supported by indirect or open to alternative interpretation evidence, and it is still unknown whether endosome localized GPCRs are even present in an active state. The use of conformation-specific single-domain antibodies to directly probe activation of the β2-adrenoceptor, a prototypical GPCR, and its cognate G protein, Gs, in living mammalian cells is described in this article. Researchers demonstrate that the adrenergic agonist isoprenaline promotes receptor and G protein activation in the plasma membrane as expected, but also in the early endosome membrane. Internalized receptors contribute to the overall cellular cyclic AMP response shortly after agonist application. These results offer concrete evidence in favor of the idea that canonical GPCR signaling emanates from both endosomes and the plasma membrane, and they offer a flexible method for observing dynamic conformational change in vivo. |
| Result |
The crucial biochemical process that starts classical GPCR signal transduction is guanine nucleotide dissociation from the cytoplasmic GTP-binding protein Gαs, and it is stabilized by ligand binding to the extracellular surface of the β2-adrenoceptor (β2-AR). B-arrestins bind to and phosphorylate activated β2-ARs, which prevents them from interacting with G proteins and encourages the endocytosis of receptors through clathrin-coated pits (CCPs). Thus, the plasma membrane has long been thought to be the only location for acute β2-AR Gas signaling. This presumption hasn't been formally examined, though. To achieve this, researchers developed a biosensor of activated β2-AR based on a single-domain camelid antibody (Nb80) that has recently been used in structural studies. Researchers hypothesized that the single-domain antibody, which preferentially binds the agonist-occupied β2-AR and can stabilize an activated receptor conformation when present in vitro at high concentrations, might function as a sensor of receptor activation when expressed at relatively low concentrations in intact cells.
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Fig.2 Nb80-GFP detects activated β2-ARs in the plasma
membrane and endosomes. (Irannejad, 2013)
References
- Rosenbaum, D.M.; et al. The structure and function of G-protein-coupled receptors. Nature. 2009, 459(7245):356-363.
- 2.Irannejad, R.; et al. Confirmational biosensors reveal GPCR signalling from endosomes. Nature. 2013, 495(7442):534-538.
