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High-level production and characterization of a G-protein coupled receptor signaling complex

MPG-Autoren
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Marino,  Stephen F.
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society;

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Zitation

Marino, S. F. (2009). High-level production and characterization of a G-protein coupled receptor signaling complex. FEBS Journal, 276(16), 4515-4528.


Zitierlink: http://hdl.handle.net/11858/00-001M-0000-0024-D7CE-A
Zusammenfassung
Elucidation of the molecular details of signal transduction through G-protein 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 highlevel expression in mammalian cells and characterization of a fusion complex between the kappa opioid receptor and its cognate G-protein alpha subunit, Gαi1. Optimization of growth conditions resulted in the highest level of active binding sites reported to date for either opioid receptors or GPCR–Gα 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 Gα 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 producing welldiffracting crystals from the purified complex