Real-time analysis of G protein-coupled receptor reconstitution in a solubilized system

The Journal of Biological Chemistry
T A BennettL A Sklar

Abstract

Receptor based signaling mechanisms are the primary source of cellular regulation. The superfamily of G protein-coupled receptors is the largest and most ubiquitous of the receptor mediated processes. We describe here the analysis in real-time of the assembly and disassembly of soluble G protein-coupled receptor-G protein complexes. A fluorometric method was utilized to determine the dissociation of a fluorescent ligand from the receptor solubilized in detergent. The ligand dissociation rate differs between a receptor coupled to a G protein and the receptor alone. By observing the sensitivity of the dissociation of a fluorescent ligand to the presence of guanine nucleotide, we have shown a time- and concentration-dependent reconstitution of the N-formyl peptide receptor with endogenous G proteins. Furthermore, after the clearing of endogenous G proteins, purified Galpha subunits premixed with bovine brain Gbetagamma subunits were also able to reconstitute with the solubilized receptors. The solubilized N-formyl peptide receptor and Galpha(i3) protein interacted with an affinity of approximately 10(-6) m with other alpha subunits exhibiting lower affinities (Galpha(i3) > Galpha(i2) > Galpha(i1) Galpha(o)). The N-formyl peptide r...Continue Reading

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Citations

Jan 14, 2003·Biophysical Journal·Xiaoqin HuangKaixian Chen
Sep 14, 2011·Journal of the American Chemical Society·Sophie RoizardHorst Vogel
Nov 9, 2004·Trends in Pharmacological Sciences·Anna WallerLarry A Sklar
Feb 8, 2006·Pharmacology & Therapeutics·Vsevolod V Gurevich, Eugenia V Gurevich
May 4, 2002·Annual Review of Biophysics and Biomolecular Structure·Larry A SklarEric R Prossnitz
Oct 20, 2005·Journal of Biomolecular Screening·Wayne R LeifertEdward J McMurchie
Nov 9, 2002·The Journal of Biological Chemistry·T Alexander KeyEric R Prossnitz
Apr 11, 2006·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·John P Nolan, Francis Mandy
Dec 24, 2005·Molecular Membrane Biology·Wayne R LeifertEdward J McMurchie
Aug 7, 2003·The Journal of Immunology : Official Journal of the American Association of Immunologists·Scott E VanCompernolleScott C Todd

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