TAILIEUCHUNG - Báo cáo khoa học: High-level production and characterization of a G-protein coupled receptor signaling complex

Elucidation of the molecular details of signal transduction through G-pro-tein coupled receptors (GPCRs) awaits the solution of high-resolution structures of the receptor species involved in passing the extracellular infor-mation across the plasma membrane. The critical challenge in this effort is the production of sufficient quantities of active and homogeneous receptor species amenable to crystallization screening. | ỊFEBS Journal High-level production and characterization of a G-protein coupled receptor signaling complex Stephen F. Marino Department of Molecular Membrane Biology Max Planck Institute of Biophysics Frankfurt am Main Germany Keywords GPCR GPCR-Ga-fusion Ga 1 kappa opioid receptor Semliki Forest Virus Correspondence S. F. Marino Department of Neurochemistry Max Planck Institute of Brain Research Deutschordenstrasse 46 D60528 Frankfurt am Main Germany Fax 49 69 96769 441 Tel 49 69 96769 262 E-mail Website http Received 5 April 2009 revised 12 June 2009 accepted 16 June 2009 doi Elucidation of the molecular details of signal transduction through G-pro-tein coupled receptors GPCRs awaits the solution of high-resolution structures of the receptor species involved in passing the extracellular information across the plasma membrane. The critical challenge in this effort is the production of sufficient quantities of active and homogeneous receptor species amenable to crystallization screening. We describe here the high-level expression in mammalian cells and characterization of a fusion complex between the kappa opioid receptor and its cognate G-protein alpha subunit Gai1. Optimization of growth conditions resulted in the highest level of active binding sites reported to date for either opioid receptors or GPCR-Ga fusions. In cells the kappa opioid receptor was stabilized against proteolysis in the context of the fusion protein and was competent to bind both agonists and antagonists. Coupling of the kappa opioid receptor with the Ga subunit was demonstrated by changes in agonist affinity in the presence of guanine nucleotides and by agonist-induced increases in the rate of guanine nucleotide hydrolysis. In addition to representing a physiologically relevant signaling complex the additional hydrophilic surface area provided by the G-protein may enhance the chances of .

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