Functional studies with membrane-bound and detergent-solubilized alpha2-adrenergic receptors expressed in Sf9 cells
| Autor: | Sercan Sen, Adrian Goldman, Päivi Pirilä, Veli-Pekka Jaakola, Moshe Finel |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Spodoptera frugiperda Sf9 cell
Insecta Time Factors G protein Recombinant Fusion Proteins Blotting Western Detergents Green Fluorescent Proteins Molecular Sequence Data Biophysics Biosensing Techniques Biology Spodoptera Ligands Biochemistry Models Biological Cell Line Receptors G-Protein-Coupled 03 medical and health sciences Receptors Adrenergic alpha-2 Animals Humans G protein-coupled receptor Baculovirus Surface plasmon resonance Receptor 030304 developmental biology DNA Primers 0303 health sciences Base Sequence Drug discovery 030302 biochemistry & molecular biology Cell Membrane Antibodies Monoclonal Cell Biology Surface Plasmon Resonance Small molecule Fusion protein Lipids Recombinant Proteins Protein Structure Tertiary Kinetics Over-expression Localization Baculoviridae Intracellular Protein Binding |
| Zdroj: | Biochimica et biophysica acta. 1712(1) |
| ISSN: | 0006-3002 |
| Popis: | A chip-based biosensor technology using surface plasmon resonance (SPR) was developed for studying the interaction of ligands and G protein-coupled receptors (GPCRs). GPCRs, the fourth largest superfamily in the human genome, are the largest class of targets for drug discovery. We have expressed the three subtypes of alpha(2)-adrenergic receptor (alpha(2)-AR), a prototypical GPCR as functional fusion proteins in baculovirus-infected insect cells. The localization of the expressed receptor was observed in intracellular organelles, as detected by eGFP fluorescence. In addition, the deletion mutants of alpha(2B)-AR, with a deletion in the 3rd intracellular loop, exhibited unaltered K(d) values and enhanced stability, thus making them more promising candidates for crystallization. SPR demonstrated that small molecule ligands can bind the detergent-solubilized receptor, thus proving that alpha(2)-AR is active even in a lipid-free environment. The K(d) values obtained from the biosensor analysis and traditional ligand binding studies correlate well with each other. This is the first demonstration of the binding of a small molecule to the detergent-solubilized state of alpha(2)-ARs and interaction of low-molecular mass-ligands in real time in a label-free environment. This technology will also allow the development of high throughput platform for screening a large number of compounds for generation of leads. |
| Databáze: | OpenAIRE |
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